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Transcription and translation

Genes provide information for building proteins . They don’t however directly create proteins. The production of proteins is completed through two processes: transcription and translation.

Transcription and translation take the information in DNA and use it to produce proteins. Transcription uses a strand of DNA as a template to build a molecule called RNA.

The RNA molecule is the link between DNA and the production of proteins. During translation, the RNA molecule created in the transcription process delivers information from the DNA to the protein-building machines.

DNA → RNA → Protein

DNA and RNA are similar molecules and are both built from smaller molecules called nucleotides. Proteins are made from a sequence of amino acids rather than nucleotides. Transcription and translation are the two processes that convert a sequence of nucleotides from DNA into a sequence of amino acids to build the desired protein.

These two processes are essential for life. They are found in all organisms – eukaryotic and prokaryotic . Converting genetic information into proteins has kept life in existence for billions of years.

DNA and RNA

RNA and DNA are very similar molecules. They are both nucleic acids (one of the four  molecules of life ), they are both built on a foundation of nucleotides and they both contain four nitrogenous bases that pair up.

A strand of DNA contains a chain of connecting nucleotides. Each nucleotide contains a sugar, and a nitrogenous base and a phosphate group. There is a total of four different nitrogenous bases in DNA: adenine (A), thymine (T), guanine (G), and cytosine (C).

A strand of DNA is almost always found bonded to another strand of DNA in a double helix. Two strands of DNA are bonded together by their nitrogenous bases. The bases form what are called ‘base pairs’ where adenine and thymine bond together and guanine and cytosine bond together.

Adenine and thymine are complementary bases and do not bond with the guanine and cytosine. Guanine and cytosine only bond with each other and not adenine or thymine.

There are a couple of key differences between the structure of DNA and RNA molecules. They contain different sugars. DNA has a deoxyribose sugar while RNA has a ribose sugar.

While three of their four nitrogenous bases are the same, RNA molecules the have a base called uracil (U) instead of a thymine base. During transcription, uracil replaces the position of thymine and forms complementary pairs with adenine.

Transcription

Transcription is the process of producing a strand of RNA from a strand of DNA. Similar to the way DNA is used as a template in DNA replication , it is again used as a template during transcription. The information that is stored in DNA molecules is rewritten or ‘transcribed’ into a new RNA molecule.

Sequence of nitrogenous bases and the template strand

Each nitrogenous base of a DNA molecule provides a piece of information for protein production. A strand of DNA has a specific sequence of bases. The specific sequence provides the information for the production of a specific protein.

Through transcription, the sequence of bases of the DNA is transcribed into the reciprocal sequence of bases in a strand of RNA. Through transcription, the information of the DNA molecule is passed onto the new strand of RNA which can then carry the information to where proteins are produced. RNA molecules used for this purpose are known as messenger RNA (mRNA).

A gene is a particular segment of DNA. The sequence of bases in for a gene determines the sequence of nucleotides along an RNA molecule.

Only one strand of a DNA double helix is transcribed for each gene. This strand is known as the ‘template strand’. The same template strand of DNA is used every time that particular gene is transcribed. The opposite strand of the DNA double helix may be transcribed for other genes.

RNA polymerase

An enzyme called ‘RNA polymerase’ is responsible for separating the two strands of DNA in a double helix. As it separates the two strands, RNA polymerase builds a strand of mRNA by adding the complementary nucleotides (A, U, G, C) to the template strand of DNA.

A specific set of nucleotides along the template strand of DNA indicates where the gene starts and where the RNA polymerase should attach and begin unravelling the double helix. The section of DNA or the gene that is transcribed is known as the ‘transcription unit’.

Rather than RNA polymerase moving along the DNA strand, the DNA moves through the RNA polymerase enzyme. As the template strand moves through the enzyme, it is unravelled and RNA nucleotides are added to the growing mRNA molecule.

As the RNA molecule grows it is separated from the template strand. The DNA template strand reforms the bonds with its complementary DNA strand to reform a double helix.

In prokaryotic cells, such as bacteria , once a specific sequence of nucleotides has been transcribed then transcription is completed. This specific sequence of nucleotides is called the ‘terminator sequence’.

Once the terminator sequence is transcribed, RNA polymerase detaches from the DNA template strand and releases the RNA molecule. No further modifications are required for the mRNA molecule and it is possible for translation to begin immediately. Translation can begin in bacteria while transcription is still occurring.

Modification of mRNA in eukaryotic cells

Creating a completed mRNA molecule isn’t quite as simple in eukaryotic cells. Like prokaryotic cells, the end of a transcription unit is signalled by a certain sequence of nucleotides. Unlike prokaryotic cells, however, RNA polymerase continues to add nucleotides after transcribing the terminator sequence.

Proteins are required to release the RNA polymerase from the template DNA strand and the RNA molecule is modified to remove the extra nucleotides along with certain unwanted sections of the RNA strand. The remaining sections are spliced together and the final mRNA strand is ready for translation.

In eukaryotic cells, transcription of a DNA strand must be complete before translation can begin. The two processes are separated by the membrane of the nucleus so they cannot be performed on the same strand at the same time as they are in prokaryotic cells.

Rate of transcription

If a certain protein is required in large numbers, one gene can be transcribed by several RNA polymerase enzymes at one time. This makes it possible for a large number of proteins to be produced from multiple RNA molecules in a short time.

Translation

Translation is the process where the information carried in mRNA molecules is used to create proteins. The specific sequence of nucleotides in the mRNA molecule provides the code for the production of a protein with a specific sequence of amino acids.

Much like how RNA is built from many nucleotides, a protein is formed from many amino acids. A chain of amino acids is called a ‘polypeptide chain’ and a polypeptide chain bends and folds on itself to form a protein.

During translation, the information of the strand of RNA is ‘translated’ from RNA language into polypeptide language i.e. the sequence of nucleotides is translated into a sequence of amino acids.

Translation occurs in ribosomes

Ribosomes are small cellular machines that control the production of proteins in cells. They are made from proteins and RNA molecules and provide a platform for mRNA molecules to couple with complimentary transfer RNA (tRNA) molecules.

Each tRNA molecule is bound to an amino acid and delivers the necessary amino acid to the ribosome. The tRNA molecules bind to the complementary bases of the mRNA molecule.

The bonded mRNA and tRNA are fed through the ribosome and the amino acid attached to the tRNA molecule is added to the growing polypeptide chain as it moves through the ribosome.

Nucleotide bases are translated into 20 different amino acids

RNA molecules only contain four different types of nitrogenous bases but there are 20 different amino acids that are used to build proteins. In order to turn four into 20, a combination of three nitrogenous bases provides the information for one amino acid.

A strand of mRNA obviously has multiple codons which provide the information for multiple amino acids. A tRNA molecule reads along one codon of the mRNA strand and collects the necessary amino acid from the cytoplasm.

The tRNA returns to the ribosome with the amino acid, binds to the complementary bases of the mRNA codon, and the amino acid is added to the end of polypeptide chain as the RNA molecules move through the ribosome.

There is a different tRNA molecule for each of the different codons of the mRNA strand. Each tRNA molecule contains three nitrogenous bases that are complementary to the three bases of a codon on the mRNA strand.

The three bases of the tRNA molecule are known as an anticodon. For example, an mRNA codon with bases UGU would have a complementary tRNA with an anticodon AGA.

The opposite end of the tRNA molecule has a site where a specific amino acid can bind to. When the tRNA recognises its complementary codon in the mRNA strand, it goes to collects its specific amino acid. The amino acid is bonded to the tRNA molecule by enzymes in the cytoplasm.

As the tRNA molecule returns with the amino acid, the anticodon of the tRNA binds to the codon of the mRNA and moves through the ribosome. Each tRNA molecule can collect and deliver multiple amino acids. One codon at a time, amino acids are brought to the ribosome and the polypeptide chain is built.

Ribosome binding sites

Ribosomes have three sites for different stages of interaction with tRNA and mRNA: the P site, A site and E site. The P site is where the ribosome holds the polypeptide chain and where the tRNA adds its amino acid to the growing chain.

The A site is where tRNA molecules bind to the codons of the mRNA strand and the E site or exit site is where the tRNA is released from the ribosome and the mRNA strand.

Translation begins when a ribosome binds to an mRNA strand and an initiator tRNA. The initiator tRNA delivers an amino acid called ‘methionine’ directly to the P site and keeps the A site open for the second tRNA molecule to bind to.

The strand of mRNA moves through the ribosome from the A site to the P site and exits at the E site. Molecules of tRNA bind to the codons of the mRNA at the A site before moving to the P site where their amino acid is attached to the end of the growing polypeptide chain.

Once tRNA molecules have released their amino acids they move into the E site and are released from the mRNA and ribosome. As one tRNA molecule moves from the P site into the E site another tRNA molecule moves from the A site into the P site and delivers the next amino acid to the polypeptide chain.

Termination of translation and modification of the polypeptide

Translation ends when a stop codon on the mRNA strand reaches the A site in the ribosome. The stop codon doesn’t have a complementary tRNA or anticodon.

Instead, a protein called a ‘release factor’ binds to the stop codon and adds a water molecule to the polypeptide chain when it moves into the P site. Once the water molecule is added to the polypeptide, the polypeptide is released from the ribosome.

It is common for multiple strands of mRNA to be translated simultaneously by multiple ribosomes. This greatly increases the rate of protein production.

A polypeptide chain must fold on itself to create its final shape as a protein. As the polypeptide is being made it is already folding into a protein. Other proteins are used to guide the polypeptide to fold into the correct shape.

Often a polypeptide chain will need to be modified before it is able to perform properly. A range of molecules, such as sugars and lipids , can be added to the polypeptide. Likewise, the polypeptide chain may be split into smaller chains or have amino acids removed.

Last edited: 31 August 2020

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11 Transcription and Translation

Learning Objectives

• Describe the flow of information through cells (“the central dogma”) and the cell components that participate.
• Describe the structure and potential products of a gene (polypeptide, rRNA, tRNA, mRNA) and the types of proteins required for transcription (RNA polymerases, transcription factors, etc.).
• Describe the structure of mRNA, including the 5ʼ cap and poly(A) tail.
• Summarize the processing of a pre-mRNA to mature RNA, including the splicing process (introns and exons).
• Describe the properties of the genetic code and codon assignments.
• Define the role of tRNAs in decoding the genetic code.
• Summarize the steps in all stages of translation: tRNA charging, initiation, elongation, and termination.

About this Chapter

The flow of genetic information in cells from DNA to mRNA to protein is described by the central dogma, which states that genes specify the sequence of mRNAs, which in turn specify the sequence of amino acids making up all proteins. The decoding of one molecule to another is performed by specific proteins and RNAs. Because the information stored in DNA is so central to cellular function, it makes intuitive sense that the cell would make mRNA copies of this information for protein synthesis, while keeping the DNA itself intact and protected. The copying of DNA to RNA is relatively straightforward, with one nucleotide being added to the mRNA strand for every nucleotide read in the DNA strand.

The translation to protein is a bit more complex because three mRNA nucleotides correspond to one amino acid in the polypeptide sequence. However, the translation to protein is still systematic and colinear.

11.1 Transcription

Unlike DNA synthesis, which only occurs during the S phase of the cell cycle, transcription and translation are continuous processes within the cell. The 5ʼ to 3ʼ strand of a DNA sequence functions as the coding ( nontemplate ) strand for the process of transcription such that the transcribed product will be identical to the coding strand, except for the insertion of uracil for thymidine (figure 11.1). The transcribed mRNA will serve as the template for protein translation.

Gene structure

The chromosome is organized into functional units call genes. These are specific locations on a chromosome that are composed of a transcribed region and a regulatory (or promoter) region. The transcribed region is typically (but not always) downstream of the transcriptional start and contains the following DNA elements: a 5ʼ cap site (required for maturation of mRNA), translational start (AUG), introns and exons, and the polyadenylation site (figure 11.2).

The regulatory or promoter region is upstream of the transcriptional start and contains regulatory elements such as:

• TATA box, which provides an accessible region for the DNA to begin to unwind, allowing for access by the transcriptional machinery, and
• CAAT or GC box and enhancers or repressors (for eukaryotic transcription), which help modulate the amount of transcript produced in any given cell.

In eukaryotes, a single gene will produce one gene product as all genes are regulated independently. This is in contrast to prokaryotes, which regulate genes in an operon structure where one mRNA may be polycistronic and encode for multiple protein products.

Types of RNA polymerase

RNA polymerase I is located in the nucleolus, a specialized nuclear substructure in which ribosomal RNA (rRNA) is transcribed, processed, and assembled into ribosomes. RNA polymerase I synthesizes all the rRNAs from the tandemly duplicated set of 18S , 5.8S, and 28S ribosomal genes. (Note that the “S” designation applies to “Svedberg” units, a nonadditive value that characterizes the speed at which a particle sediments during centrifugation.)

RNA polymerase II is located in the nucleus and synthesizes all protein-coding nuclear pre-mRNAs. Eukaryotic pre-mRNAs undergo extensive processing after transcription but before translation.

RNA polymerase II is responsible for transcribing the overwhelming majority of eukaryotic genes. RNA polymerase III is also located in the nucleus. This polymerase transcribes a variety of structural RNAs that includes the 5S pre-rRNA, transfer pre-RNAs (pre-tRNAs), and small nuclear pre-RNAs. The tRNAs have a critical role in translation; they serve as the “adaptor molecules” between the mRNA template and the growing polypeptide chain. Small nuclear RNAs have a variety of functions, including “splicing” pre-mRNAs and regulating transcription factors.

Locations, products, and sensitivities of the three eukaryotic RNA polymerases

Table 11.1: Locations, products, and sensitivities of the three eukaryotic RNA polymerases.

Transcription

Eukaryotes assemble a complex of transcription factors required to recruit RNA polymerase II to a protein coding gene.

Transcription factors that bind to the promoter are called basal transcription factors. These basal factors are all called TFII (for transcription factor/polymerase II) plus an additional letter (A–J). The core complex is TFIID, which includes a TATA-binding protein (TBP). The other transcription factors systematically fall into place on the DNA template, with each one further stabilizing the pre-initiation complex and contributing to the recruitment of RNA polymerase II (figure 11.3).

Some eukaryotic promoters also have a conserved CAAT box (GGCCAATCT) at approximately -80. Further upstream of the TATA box, eukaryotic promoters may also contain one or more GC-rich boxes (GGCG) or octamer boxes (ATTTGCAT). These elements bind cellular factors that increase the efficiency of transcription initiation and are often identified in more “active” genes that are constantly being expressed by the cell. Other regulatory elements within the promoter region will be discussed in section 12.1 .

Following the formation of the pre-initiation complex, the polymerase is released from the other transcription factors, and elongation is allowed to proceed with the polymerase synthesizing pre-mRNA in the 5′ to 3′ direction.

Termination

The termination of transcription is different for the different polymerases. Unlike in prokaryotes, elongation by RNA polymerase II in eukaryotes takes place 1,000 to 2,000 nucleotides beyond the end of the gene being transcribed. This pre-mRNA tail is subsequently removed by cleavage during mRNA processing. Alternatively, RNA polymerases I and III require termination signals. Genes transcribed by RNA polymerase I contain a specific eighteen-nucleotide sequence that is recognized by a termination protein. The process of termination in RNA polymerase III involves an mRNA hairpin similar to rho-independent termination of transcription in prokaryotes.

Types of RNA

RNA is found in three different forms in the cell, and each is used for specific aspects of translation. Not all RNA that is transcribed is translated into a protein product; some transcribed RNA (rRNA and tRNA) is fully functional in the RNA form. mRNA (messenger RNA) is transcribed by RNA pol II.

In eukaryotes, pre-mRNA requires maturation before use in translation including (figure 11.4):

• 5ʼ Capping by the addition of a 7-methylguanosine cap. Capping, resulting in the addition of two methyl groups on the 5ʼ end, is fundamental for both mRNA stabilization and for translational initiation.
• Addition of a poly(A) tail. The addition of the poly(A) tail also provides mRNA stability and is important for transcriptional termination. Neither the cap nor tail are part of the DNA coding regions.
• Splicing. Splicing involves removal of introns (noncoding regions) and retention of exons (coding regions).

Splicing is a complex process mediated by a large protein RNA-associated complex called the spliceosome. The structure contains both proteins and small nuclear (sn)RNA. (Note antibodies to snRNAs are specific for systemic lupus.) Intronic sequences usually have GU at their 5′ end and AG at their 3′ end. An adenosine (A) is typically found at the branching point within the intron sequence. Small nuclear ribonucleoproteins (snRNPs) of the spliceosome recognize intron‒exon junctions and splice out the intron as a “lariat” structure. Splicing starts with an autocatalytic cleavage of the 5ʼ end of the intron leading to the formation of a circular or lariat where a 5′ UG sequence pairs with an internal adenine (A) or branch site. Finally the 3ʼ end of the intron is cleaved, and the intron is released as a lariat, and the right side of the exon is spliced to the left side. Alternative splicing of introns and exons generates protein variation from a single mRNA (figure 11.5).

tRNA, transfer RNA, is transcribed by RNA pol III, and like mRNA it requires maturation including:

• Removal of introns,
• The addition of the 3ʼ amino acid attachment site (CCA), and
• Folding into a clover like structure.

tRNAs also are typical of base modifications generating nonconventional bases allowing base-pairing to several codons. This duplicity of binding is usually due to wobble in the third base pair. tRNA primarily functions to bring amino acids to the ribosome during protein translation. The anticodon on tRNA pairs with the codon on mRNA, and this determines which amino acid is added to the growing polypeptide chain.

rRNA, ribosomal RNA, is transcribed by RNA poly I and III and requires maturation that is slightly different from mRNA and tRNA. This RNA product is not translated but rather requires methylation and is incorporated into the protein as structural support. The 18S RNA is incorporated into the 40S ribosomal subunit, and the 28S, 5.8S, and 5S is incorporated into the 60S ribosomal subunit. These combine to make the full 80S ribosome required for protein translation.

11.1 References and resources

Clark, M. A. Biology , 2nd ed. Houston, TX: OpenStax College, Rice University, 2018, Chapter 15: Genes and Proteins.

Karp, G., and J. G. Patton. Cell and Molecular Biology: Concepts and Experiments , 7th ed. Hoboken, NJ: John Wiley, 2013, Chapter 11: Gene Expression: From Transcription to Translation.

Le, T., and V. Bhushan. First Aid for the USMLE Step 1 , 29th ed. New York: McGraw Hill Education, 2018, 39, 41–45.

Nussbaum, R. L., R. R. McInnes, H. F. Willard, A. Hamosh, and M. W. Thompson. Thompson & Thompson Genetics  in Medicine , 8th ed. Philadelphia: Saunders/Elsevier, 2016, Chapter 3: The Human Genome: Gene Structure and Function.

Grey, Kindred, Figure 11.3 Transcription initiation. 2021. https://archive.org/details/11.3_20210926. CC BY 4.0 .

Grey, Kindred, Figure 11.4 Overview of mRNA processing involving the removal of introns (splicing), addition of a 5’ cap and 3’ tail. 2021. https://archive.org/details/11.4_20210926. CC BY 4.0 .

Grey, Kindred, Figure 11.5 Summary of mRNA splicing. 2021. https://archive.org/details/11.5_20210926. CC BY 4.0 .

Lieberman M, Peet A. Figure 11.1 Co-linearity of DNA and RNA. Adapted under Fair Use from Marks’ Basic Medical Biochemistry. 5th Ed. pp 277. Figure 15.3 Reading frame of messenger RNA (mRNA). 2017.

Lieberman M, Peet A. Figure 11.2 Schematic view of a eukaryotic gene structure. Adapted under Fair Use from Marks’ Basic Medical Biochemistry. 5th Ed. pp 255. Figure 14.4 A schematic view of a eukarytoic gene, and steps required to produce a protein product. 2017. Added Myoglobin by AzaToth. Public domain. From Wikimedia Commons .

11.2 Protein Translation

Translation is the process by which mRNAs are converted into protein products through the interactions of mRNA, tRNA, and rRNA. Even before an mRNA is translated, a cell must invest energy to build each of its ribosomes, a complex macromolecule composed of structural and catalytic rRNAs, and many distinct polypeptides. In eukaryotes, the nucleolus is completely specialized for the synthesis and assembly of rRNAs.

Ribosomes exist in the cytoplasm and rough endoplasmic reticulum of eukaryotes. Ribosomes dissociate into large and small subunits when they are not synthesizing proteins and reassociate during the initiation of translation.

• In E. coli , the small subunit is described as 30S , and the large subunit is 50S , for a total of 70S (recall that Svedberg units are not additive).
• Mammalian ribosomes have a small 40S subunit and a large 60S subunit, for a total of 80S . The small subunit is responsible for binding the mRNA template, whereas the large subunit sequentially binds tRNAs.

Each mRNA molecule is simultaneously translated by many ribosomes, all synthesizing protein in the same direction: reading the mRNA from 5′ to 3′ and synthesizing the polypeptide from the N terminus to the C terminus. The complete mRNA/poly-ribosome structure is called a polysome.

tRNA synthetases

mRNAs are read three base pairs at a time (codon), and the reading frame will start with the first AUG (figures 11.6 and 11.7). Translation requires the formation of an aminoacyl-tRNA where tRNA is charged with the correct amino acid and brought to the translational machinery. Through the process of tRNA “charging,” each tRNA molecule is linked to its correct amino acid by one of a group of enzymes called aminoacyl tRNA synthetases.

At least one type of aminoacyl tRNA synthetase exists for each of the twenty amino acids; the exact number of aminoacyl tRNA synthetases varies by species. These enzymes first bind and hydrolyze ATP to catalyze a high-energy bond between an amino acid and adenosine monophosphate (AMP). The activated amino acid is then transferred to the tRNA, and AMP is released. The term “charging” is appropriate, since the high-energy bond that attaches an amino acid to its tRNA is later used to drive the formation of the peptide bond. Each tRNA is named for its amino acid.

Translational initiation

Translation is initiated by the assembly of the small ribosomal subunit ( 40S ) with initiation factors (IF), which recognize the 5ʼ cap of the mRNA. This is referred to as the cap-binding complex, and this will scan the mRNA for the initial AUG needed to start translation. Once at the cap, the initiation complex tracks along the mRNA in the 5′ to 3′ direction, searching for the AUG start codon. Many eukaryotic mRNAs are translated from the first AUG, but this is not always the case. Once the appropriate AUG is identified, the other proteins and CBP dissociate, and the 60S subunit binds to the complex of Met- tRNAi , mRNA, and the 40S subunit. This step completes the initiation of translation in eukaryotes (figure 11.8).

Translation elongation

The ribosome has three locations for tRNA binding: A, P, and E sites.

• All tRNAs enter into the A site except for the initial methionine tRNA, which binds to the P site.
• The initial tRNA carrying methionine will attach to the ribosomal P site, and GTP is hydrolyzed, leading to the release of IF factors and recruitment of the large ribosomal subunit forming the complete ribosome.
• All tRNAs exit the ribosome through the E site.

Translation elongation requires energy in the form of GTP, and additional elongation factors (EFs) are required for this process. Elongation proceeds with charged tRNAs sequentially entering and leaving the ribosome as each new amino acid is added to the polypeptide chain. Movement of a tRNA from A to P to E sites is induced by conformational changes that advance the ribosome by three bases in the 3′ direction. GTP energy is required both for the binding of a new aminoacyl-tRNA to the A site and for its translocation to the P site after formation of the peptide bond.

Peptide bonds form between the amino group of the amino acid attached to the A-site tRNA and the carboxyl group of the amino acid attached to the P-site tRNA. A new tRNA with the corresponding amino acid coded for by the mRNA will enter into the A site of the ribosome.

The amino acid attached to the tRNA in the P site will be transferred to the tRNA in the A site; this is referred to as the peptidyl transferase react ion. The tRNAs will slide such that the tRNA in the P site will move to the E site and the tRNA in the A site will move to the P site. The tRNA in the E site will be released, and a new tRNA will enter into the A site, and the process will continue with the addition of tRNAs in the manner until the full message is transcribed (figure 11.8).

Translational termination

Termination of translation occurs when a nonsense codon (UAA, UAG, or UGA) is encountered. Upon aligning with the A site, these nonsense codons are recognized by protein release factors that resemble tRNAs.

The release factors in both prokaryotes and eukaryotes instruct peptidyl transferase to add a water molecule to the carboxyl end of the P-site amino acid. This reaction forces the P-site amino acid to detach from its tRNA, and the newly made protein is released.

The small and large ribosomal subunits dissociate from the mRNA and from each other; they are recruited almost immediately into another translation initiation complex. After many ribosomes have completed translation, the mRNA is degraded so the nucleotides can be reused in another transcription reaction.

11.2 References and resources

Grey, Kindred, Figure 11.6 Genetic code, each codons is 3 nucleotides corresponding to a specific amino acid. The code is degenerate meaning several codes are present for the same amino acid and the codes for similar amino acids are clustered. 2021. https://archive.org/details/11.6_20210926. CC BY 4.0 .

Grey, Kindred, Figure 11.7: Summary of translational initiation. 2021. CC BY SA 3.0 . Adapted from Eukaryotic Translation Initiation by Chewie. CC BY SA 3.0 . From Wikimedia Commons .

Grey, Kindred, Figure 11.8 Summary of translational elongation. 2021. CC BY 4.0 .

Cell Biology, Genetics, and Biochemistry for Pre-Clinical Students Copyright © 2021 by Renée J. LeClair is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License , except where otherwise noted.

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Translation: DNA to mRNA to Protein

The genes in DNA encode protein molecules, which are the "workhorses" of the cell , carrying out all the functions necessary for life. For example, enzymes, including those that metabolize nutrients and synthesize new cellular constituents, as well as DNA polymerases and other enzymes that make copies of DNA during cell division , are all proteins.

In the simplest sense, expressing a gene means manufacturing its corresponding protein, and this multilayered process has two major steps. In the first step, the information in DNA is transferred to a messenger RNA ( mRNA ) molecule by way of a process called transcription . During transcription , the DNA of a gene serves as a template for complementary base-pairing , and an enzyme called RNA polymerase II catalyzes the formation of a pre-mRNA molecule, which is then processed to form mature mRNA (Figure 1). The resulting mRNA is a single-stranded copy of the gene, which next must be translated into a protein molecule.

Where Translation Occurs

Within all cells, the translation machinery resides within a specialized organelle called the ribosome . In eukaryotes, mature mRNA molecules must leave the nucleus and travel to the cytoplasm , where the ribosomes are located. On the other hand, in prokaryotic organisms, ribosomes can attach to mRNA while it is still being transcribed. In this situation, translation begins at the 5' end of the mRNA while the 3' end is still attached to DNA.

In all types of cells, the ribosome is composed of two subunits: the large (50S) subunit and the small (30S) subunit (S, for svedberg unit, is a measure of sedimentation velocity and, therefore, mass). Each subunit exists separately in the cytoplasm, but the two join together on the mRNA molecule. The ribosomal subunits contain proteins and specialized RNA molecules—specifically, ribosomal RNA ( rRNA ) and transfer RNA ( tRNA ) . The tRNA molecules are adaptor molecules—they have one end that can read the triplet code in the mRNA through complementary base-pairing, and another end that attaches to a specific amino acid (Chapeville et al. , 1962; Grunberger et al. , 1969). The idea that tRNA was an adaptor molecule was first proposed by Francis Crick, co-discoverer of DNA structure, who did much of the key work in deciphering the genetic code (Crick, 1958).

Within the ribosome, the mRNA and aminoacyl-tRNA complexes are held together closely, which facilitates base-pairing. The rRNA catalyzes the attachment of each new amino acid to the growing chain.

The Beginning of mRNA Is Not Translated

Interestingly, not all regions of an mRNA molecule correspond to particular amino acids. In particular, there is an area near the 5' end of the molecule that is known as the untranslated region (UTR) or leader sequence. This portion of mRNA is located between the first nucleotide that is transcribed and the start codon (AUG) of the coding region, and it does not affect the sequence of amino acids in a protein (Figure 3).

So, what is the purpose of the UTR? It turns out that the leader sequence is important because it contains a ribosome-binding site. In bacteria , this site is known as the Shine-Dalgarno box (AGGAGG), after scientists John Shine and Lynn Dalgarno, who first characterized it. A similar site in vertebrates was characterized by Marilyn Kozak and is thus known as the Kozak box. In bacterial mRNA, the 5' UTR is normally short; in human mRNA, the median length of the 5' UTR is about 170 nucleotides. If the leader is long, it may contain regulatory sequences, including binding sites for proteins, that can affect the stability of the mRNA or the efficiency of its translation.

Translation Begins After the Assembly of a Complex Structure

Table 1 shows the N-terminal sequences of proteins in prokaryotes and eukaryotes, based on a sample of 170 prokaryotic and 120 eukaryotic proteins (Flinta et al. , 1986). In the table, M represents methionine, A represents alanine, K represents lysine, S represents serine, and T represents threonine.

Table 1: N-Terminal Sequences of Proteins

* Methionine was removed in all of these proteins

** Methionine was not removed from any of these proteins

Once the initiation complex is formed on the mRNA, the large ribosomal subunit binds to this complex, which causes the release of IFs (initiation factors). The large subunit of the ribosome has three sites at which tRNA molecules can bind. The A (amino acid) site is the location at which the aminoacyl-tRNA anticodon base pairs up with the mRNA codon, ensuring that correct amino acid is added to the growing polypeptide chain. The P (polypeptide) site is the location at which the amino acid is transferred from its tRNA to the growing polypeptide chain. Finally, the E (exit) site is the location at which the "empty" tRNA sits before being released back into the cytoplasm to bind another amino acid and repeat the process. The initiator methionine tRNA is the only aminoacyl-tRNA that can bind in the P site of the ribosome, and the A site is aligned with the second mRNA codon. The ribosome is thus ready to bind the second aminoacyl-tRNA at the A site, which will be joined to the initiator methionine by the first peptide bond (Figure 5).

The Elongation Phase

Next, peptide bonds between the now-adjacent first and second amino acids are formed through a peptidyl transferase activity. For many years, it was thought that an enzyme catalyzed this step, but recent evidence indicates that the transferase activity is a catalytic function of rRNA (Pierce, 2000). After the peptide bond is formed, the ribosome shifts, or translocates, again, thus causing the tRNA to occupy the E site. The tRNA is then released to the cytoplasm to pick up another amino acid. In addition, the A site is now empty and ready to receive the tRNA for the next codon.

This process is repeated until all the codons in the mRNA have been read by tRNA molecules, and the amino acids attached to the tRNAs have been linked together in the growing polypeptide chain in the appropriate order. At this point, translation must be terminated, and the nascent protein must be released from the mRNA and ribosome.

Termination of Translation

There are three termination codons that are employed at the end of a protein-coding sequence in mRNA: UAA, UAG, and UGA. No tRNAs recognize these codons. Thus, in the place of these tRNAs, one of several proteins, called release factors, binds and facilitates release of the mRNA from the ribosome and subsequent dissociation of the ribosome.

Comparing Eukaryotic and Prokaryotic Translation

The translation process is very similar in prokaryotes and eukaryotes. Although different elongation, initiation, and termination factors are used, the genetic code is generally identical. As previously noted, in bacteria, transcription and translation take place simultaneously, and mRNAs are relatively short-lived. In eukaryotes, however, mRNAs have highly variable half-lives, are subject to modifications, and must exit the nucleus to be translated; these multiple steps offer additional opportunities to regulate levels of protein production, and thereby fine-tune gene expression.

References and Recommended Reading

Chapeville, F., et al. On the role of soluble ribonucleic acid in coding for amino acids. Proceedings of the National Academy of Sciences 48 , 1086–1092 (1962)

Crick, F. On protein synthesis. Symposia of the Society for Experimental Biology 12 , 138–163 (1958)

Flinta, C., et al . Sequence determinants of N-terminal protein processing. European Journal of Biochemistry 154 , 193–196 (1986)

Grunberger, D., et al . Codon recognition by enzymatically mischarged valine transfer ribonucleic acid. Science 166 , 1635–1637 (1969) doi:10.1126/science.166.3913.1635

Kozak, M. Point mutations close to the AUG initiator codon affect the efficiency of translation of rat preproinsulin in vivo . Nature 308 , 241–246 (1984) doi:10.1038308241a0 ( link to article )

---. Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cell 44 , 283–292 (1986)

---. An analysis of 5'-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Research 15 , 8125–8148 (1987)

Pierce, B. A. Genetics: A conceptual approach (New York, Freeman, 2000)

Shine, J., & Dalgarno, L. Determinant of cistron specificity in bacterial ribosomes. Nature 254 , 34–38 (1975) doi:10.1038/254034a0 ( link to article )

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// There was an extra comma at the end of multiList array. $( function($){ var quizMulti = { multiList: [ { ques: “In what direction is RNA polymerized?”, ans: “5’ to 3’”, ansSel: [“3’ to 5’”, “3 to 5”, “N to C”], ansInfo: "" }] }; var options = { allRandom: false, Random: false, help: “”, showHTML: false, animationType: 0, showWrongAns: true, title: “Concept test 1”, }; $("#quizArea").jQuizMe(quizMulti, options); });

• Transcription and translation, Excerpt 2

True or False: More than one codon typically encodes each amino acid. False close True check Check

Where does translation begin, as indicated on the mRNA transcript? promoter close start codon check terminator close transcription start site close Check

If the following were a complete mRNA, which codon would be recognized as the stop codon? 5' UAAUGCUGACUAGUUAAGCCCGAGCGAA-3' UAA check UAG close UGA close Check

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• Transcription and Translation

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• The Nobel Prize in Physiology or Medicine 1968
• Transcription Animation (Basic)
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Welcome to the Visible Body Blog!

Dna and rna basics: replication, transcription, and translation.

Posted on 6/22/21 by Laura Snider

DNA (deoxyribonucleic acid) is one of the most important molecules in your body, and though around 99.9% of your DNA is the same as that of every other human , the 0.1% that’s different is what makes you genetically unique! This tiny biological structure is the ultimate instruction manual, containing the “recipes” for the proteins your body needs to develop and function. 

Today, we’re going to give you a primer on the basics of DNA. We’ll talk about its structure, how it replicates, and the role it plays in the production of proteins. 

March 2022 Update: Visible Biology is now available! Visible Biology is a visual guide to important biological concepts and processes, including DNA and chromosomes , prokaryotic vs. eukaryotic cells , monocot and dicot plant structures , blood cells , photosynthesis , and more.

The Structure of DNA: Phenomenal Biological Powers...Itty Bitty Living Space

Did you know that in the average human cell, there is about 2m (6ft) of DNA? That’s pretty impressive, considering that even the largest cells are just over 100µm in diameter. (That’s really tiny, by the way—1µm is one millionth of a meter.)

See how you can teach and learn about DNA and chromosome structure in Visible Biology . 

How is all that genetic material packed into a space way smaller than the head of a pin? The short answer is a whole lot of twisting and winding. DNA wraps around protein clusters called histones to form units called nucleosomes. These nucleosomes fold into a zig-zag patterned fiber, which then forms loops.

There are 46 separate strings of DNA in each somatic cell of the human body. Each one of these is called a chromosome. Scientists group them into 23 homologous pairs, which means that the chromosomes in each pair are similar in structure and function. The only exception to this is the 23rd pair—the sex chromosomes—in biologically male individuals. X and Y sex chromosomes only have certain regions (autosomal regions) that are homologous.  

At the molecular level, DNA has a characteristic double-helix shape, and though this wasn’t observed by scientists until mid-20th century , it has quickly become one of the most iconic shapes in all of science. 

Lesson on DNA structure from the Visible Biology YouTube series with Dr. Cindy Harley.

The sides of this twisted ladder are composed of alternating molecules of sugar (deoxyribose, to be precise) and a phosphate group. Each side is named for the direction it runs in (5’–3’ or 3’–5’). The ladder’s “steps” are composed of two nitrogenous bases, held together with hydrogen bonds.

Four nitrogenous bases—cytosine, thymine, adenine, and guanine—can be found on strands of DNA. In terms of their chemical structure, cytosine and thymine are pyrimidines and adenine and guanine are purines. Adenine and thymine (A and T) always pair together, and guanine and cytosine (G and C) always pair together. They pair this way because A and T form two hydrogen bonds with each other and G and C form three. 

At the most basic level, different sections of DNA strands (sequences of nitrogenous bases) provide instructions for the synthesis of proteins. A single section of DNA can even code for multiple proteins!

Replication: Doubling Up on DNA

Replication of a cell’s DNA occurs before a cell prepares to undergo division—either mitosis or meiosis I.

It takes place in three(ish) steps.

• DNA unwinds from the histones.
• An enzyme called DNA helicase opens up the helix structure on a segment of DNA, breaking the bonds between the nitrogenous bases. It does this in a zipper-like fashion, leaving a replication fork behind it.
• Here’s where things get funky. 
• On the 5’–3’ strand of the DNA, an enzyme called DNA polymerase slides towards the replication fork and uses the sequence of nitrogenous bases on that strand to make a new strand of DNA complementary to it (this means that its bases pair with the ones on the old strand). 
• On the 3’–5’ strand, multiple DNA polymerases match up base pairs in partial segments, moving away from the replication fork. Later, DNA ligase connects these partial strands into a new continuous segment of DNA.

Want to know something neat? When a DNA molecule replicates, each of the resulting new DNA molecules contains a strand of the original, so neither is completely “new."  Also, new histones are made at the same time the DNA replicates so that the new strands of DNA can coil around them.

Interlude: RNA vs DNA

Before we discuss transcription and translation, the two processes key to protein synthesis, we need to talk about another kind of molecule: RNA.

RNA is a lot like DNA—it’s got a sugar-phosphate backbone and contains sequences of nitrogenous bases. However, there are a couple of vital differences between RNA and DNA:

• RNA has only one nucleotide chain. It looks like only one side of the DNA ladder.
• RNA has ribose as the sugar in its backbone.
• RNA has Uracil (U) instead of thymine.
• RNA is smaller than DNA. RNA caps out at around 10,000 bases long, while DNA averages about 100 million.
• RNA can leave the nucleus. In fact, it does most of its work in the cytoplasm.

There are several different types of RNA, each with different functions, but for the purposes of this article, we’re going to focus on messenger RNA ( m RNA) and transfer RNA ( t RNA).

Making a Protein, Part 1: Transcription

Transcription is the first phase of the protein-making process, even though the actual protein synthesis doesn’t happen until the second phase. Essentially, what happens during transcription is that an m RNA “copies down” the instructions for making a protein from DNA.

First, an enzyme called RNA polymerase opens up a section of DNA and assembles a strand of m RNA by “reading” the sequence of bases on one of the strands of DNA. If there’s a C on the DNA, there will be a G on the RNA (and vice versa). If there’s a T on the DNA, there will be an A on the RNA, but if there’s an A on the DNA, there will be a U (instead of a T) on the RNA. As the RNA polymerase travels down the string of DNA, it closes the helical structure back up after it. 

Before the new m RNA can go out to deliver its protein fabrication instructions, it gets “cleaned up” by enzymes. They remove segments called introns and then splice the remaining segments, called exons, together. Exons are the sequences that actually code for proteins, so they’re the ones the m RNA needs to keep. You can think of introns like padding between the exons.

Also, remember how I mentioned that a single sequence of DNA can code for multiple proteins? Alternative splicing is the reason why: before the m RNA leaves the nucleus, its exons can be spliced together in different ways.

Lesson on transcription from the Visible Biology YouTube series with Dr. Cindy Harley.

Making a Protein, Part 2: Translation

After it’s all cleaned up and ready to go, the m RNA leaves the nucleus and goes out to fulfill its destiny: taking part in translation, the second half of protein construction. 

Lesson on translation from the Visible Biology YouTube series with Dr. Cindy Harley.

In the cytoplasm, the m RNA must interface with t RNA with the help of a ribosome. t RNA is a type of RNA that has a place to bind to free amino acids and a special sequence of three nitrogenous bases (an anticodon) that binds to the ribosome.

Ribosomes are organelles that facilitate the meeting of t RNA and m RNA. During translation, ribosomes and t RNA follow the instructions on the m RNA and assemble amino acids into proteins. 

Each ribosome is made up of two subunits (large and small). These come together at the start of translation. Ribosomal subunits can usually be found floating around in the cytoplasm, but a ribosome will dock on the rough endoplasmic reticulum if the protein it’s making needs to be put into a transport vesicle. Ribosomes also have three binding sites where t RNA can dock: the A site ( aminoacyl , first position), the P site ( peptidyl, second position) and the E site (the exit position).

Ultimately, translation has three steps: initiation, elongation, and termination. 

During initiation, the strand of mRNA forms a loop, and a small ribosomal subunit (the bottom of the ribosome) hooks onto it and finds a sequence of bases that signals it to begin transcription. This is called the start codon (AUG).

Then, a t RNA with UAC anticodon pairs with this start codon and takes up the second position (P) site of the ribosome. This t RNA carries the amino acid Methionine (Met). At this point, the large ribosomal subunit gets in position as well (it’s above the m RNA and the small subunit is below).

In the elongation phase, the fully-assembled ribosome starts to slide along the m RNA. Let’s say the next sequence of bases it encounters after the start codon is GCU. A t RNA molecule with the anticodon CGA will bind to the first position (A) site of the ribosome. The amino acid it’s carrying (alanine) forms a peptide bond with Met. Afterward, the CGA t RNA (carrying the Met-Ala chain) moves to the second position and the UAC t RNA enters the E binding site. The first position site is then ready to accept a new t RNA. This process keeps going until the ribosome gets to a “stop” codon.

Termination is pretty much what it sounds like. Upon reaching the “stop” codon, the t RNA that binds to the first position carries a protein called a release factor. The amino acid chain then breaks off from the ribosome, either going off into the cytosol or into the cisterna of the rough ER, and the ribosome disassembles. However, it might very well reassemble and go around the mRNA loop again. Also, multiple ribosomes can work on the same mRNA at once!

And those are the basics of DNA! 

Here’s a handy chart you can look at if you need to remember the differences between transcription, translation, and replication: 

If you want to learn more about cells, check out these related VB Blog posts: 

• Anatomy & Physiology: Parts of a Human Cell
• Tiny Transportation: Passive vs. Active Transport in Cells

For more information about the structure of DNA and chromosomes, check out our DNA eBook! 

Be sure to subscribe to the  Visible Body  Blog for more anatomy awesomeness! 

Are you an instructor? We have award-winning 3D products and resources for your anatomy and physiology course!  Learn more here.

Additional Sources: 

• Crash Course Biology #10: DNA Structure and Replication
• Crash Course Biology #11: DNA, Hot Pockets, & The Longest Word Ever
• Saladin (2015). Anatomy & Physiology: The Unity of Form and Function . 7th ed.
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Transcription and Translation Lesson Plan

This list of websites provide tools and resources for teaching the concepts of transcription and translation, two key steps in gene expression .

Definitions

Transcription is the process of making an RNA copy of a gene sequence. This copy, called a messenger RNA (mRNA) molecule, leaves the cell nucleus and enters the cytoplasm, where it directs the synthesis of the protein, which it encodes. Here is a more complete definition of transcription:  Transcription Translation is the process of translating the sequence of a messenger RNA (mRNA) molecule to a sequence of amino acids during protein synthesis. The genetic code describes the relationship between the sequence of base pairs in a gene and the corresponding amino acid sequence that it encodes. In the cell cytoplasm, the ribosome reads the sequence of the mRNA in groups of three bases to assemble the protein. Here is a more complete definition of translation:  Translation

Teachers' Domain: Cell Transcription and Translation

Teachers' Domain is a free educational resource produced by WGBH with funding from the NSF, which houses thousands of media resources, support materials, and tools for classroom lessons.One of these resources focuses on the topics of transcription and translation.This resource is an interactive activity that starts with a general overview of the central dogma of molecular biology, and then goes into more specific details about the processes of transcription and translation.In addition to the interactive activity, the resource also includes a background narrative and discussion questions that could be used for assessment.Although the material is designated as appropriate content for grades, 9-12, it would serve as an excellent introduction to the topic for biology majors, or would be well suited for non-biology majors at the post-secondary level. See:  Teachers' Domain: Cell Transcription and Translation  

The DNA Learning Center's (DNALC) The Howard Hughes Medical Institute's DNA interactive (DNAi) The University of Utah's Genetic Science Learning Center

The DNA Learning Center's (DNALC) website, the Howard Hughes Medical Institute's DNA interactive (DNAi) website, and the University of Utah's Genetic Science Learning Center website listed below contain excellent narrated animations describing transcription and translation. These animations are useful as a lecture supplement or for students to review on their own. The DNALC animations cover central dogma, transcription (basic and advanced), mRNA splicing, RNA splicing, triplet code and translation (basic and advanced). The DNAi modules," Reading the Code" and "Copying the Code," describe the history of the process, the scientists involved in the discovery, and the basics of the process, and also include an animation and interactive game. Particularly useful to students are the interactive animations from the University of Utah that allow one to, for example,"Transcribe/Translate a Gene"or examine the effects of gene mutation as they "Test Neurofibromin Activity in a Cell."

The DNA Learning Center's (DNALC):  3-D Animation Library The Howard Hughes Medical Institute's DNA interactive: (DNAi):  Code The University of Utah's Genetic Science Learning Center:  Transcribe and Translate a Gene

The Nature Education website, Scitable, is a great study resource for students who want to learn more about, or are having difficulty understanding, transcription and translation. The site contains a searchable library, including many "overviews" of transcription, translation, and related topics. Students have access to a Genetics "Study Pack", which provides explanations, animations, and links to other resources.In addition, Scitable has an "Ask An Expert" feature that allows students to submit specific genetics-related questions. See:  Scitable

NHGRI Talking Glossary of Genetics Terms iPhone App and Website

The Talking Glossary of Genetics Terms website and iPhone app provide an easily transportable and accessible reference for your students. Many times the unfamiliar vocabulary is the major stumbling block to student comprehension. This app/site gives them a handy reference to common terms used in describing the components involved on transcription and translation. Talking Glossary of Genetics Terms Talking Glossary of Genetics Terms iPhone App

University of Buffalo Case Study Collection: Decoding the Flu

This "clicker case" was designed to develop students' ability to read and interpret information stored in DNA. Making use of personal response systems ("clickers") along with a PowerPoint presentation, students follow the story of "Jason," a student intern at the Centers for Disease Control & Prevention (CDC). While working with a CDC team in Mexico, Jason is the only person who does not get sick from a new strain of flu. It is up to Jason to use molecular data collected from different local strains of flu to identify which one may be causing the illness. Although designed for an introductory biology course for science or non-science majors, the case could be adapted for upper-level courses by including more complex problems and aspects of gene expression, such as the excision of introns." See:  Decoding the Flu

Protein Synthesis Animation from Biology-Forums.com

Translation is the process of producing proteins from the mRNA. This YouTube video shows the molecular components involved in the process. It also animates how the peptide is elongated through interaction between mRNA, ribosome, tRNA, and residues.  Protein Synthese Animation

The Central Dogma Animation by RIKEN Omics Science Center

The 'Central Dogma' of molecular biology is that 'DNA makes RNA makes protein'. This anime shows how molecular machines transcribe the genes in the DNA of every cell into portable RNA messages, how those messenger RNA are modified and exported from the nucleus, and finally how the RNA code is read to build proteins.  Animation: The Central Dogma

A Prezi of this information can be found at:  NHGRI Teacher Resouces-Central Dogma

Contributing Team of Educators:

Kari D. Loomis, Ph.D., Mars Hill College Luisel Ricks, Ph.D., Howard University Mark Bolt, Ph.D., University of Pikeville Cathy Dobbs, Ph.D., Joliet Junior College Changhui Yan, Ph.D., North Dakota State University Solomon Adekunle, Ph.D., Southern University

Last updated: February 13, 2014

Want to create or adapt books like this? Learn more about how Pressbooks supports open publishing practices.

2 Transcription, Translation

Session level objectives (slos): after completing the session, students will be able to:.

SLO 1. Explain how cell s overc om e three m ajor c hal lenges t o replic at e their genom es.

SLO 2. Explain w hy different genes need t o be expres s ed at different rates , in d ifferent cells , a t d ifferent times .

SLO 3.   Describe   the functi ons of the three mamm alian RN A polym er as es.

SLO 4. Explain the structure o f a mammalian gene, including regulatory and coding elements.

SLO 5. Describe how RNA polymerase II (RNAP II) is directed to gene promoters and the roles of general transcription factors.

Slo 6. explain how transcription factors control which proteins are made in different cell types., slo 7. outline the processing reactions that precede export of an mrna from nucleus to cytoplasm..

SLO 8. Outline the ma j o r s te ps o f p r o tein sy nthe s is, including   the r o le s o f mRN A, t RN A, t RN A sy nthet as e s a n d   ribosom es.

SLO 9 . D escribe the bas ic ways in w hich m icr o RN A (miRNA) mol ecules c ont r ol gene expres s ion.

Common Themes of Information Transfer

DNA — replication —> DNA — transcription —> RNA — translation —> polypeptide

There are common themes in these reactions.

• Biological polymerization reactions always have an intrinsic directionality (polarity): In DNA replication and RNA transcription, the template strand is always read 3 ´- to – 5´ , and the nascent chain is always synthesized 5´- to -3´ . In protein synthesis (translation), the mRNA template is always read 5 ´- to -3´ , and the nascent polypept ide is always s ynt hesized N -to- C .
• The polymerase must be accurately positioned at a start site on the template. In each case this process is called initiation , and in each case initiation entails several regulated steps.
• The polymerase has an elongation cycle, which is what it sounds like. This is the major biosynthetic stage.
• A signal on the template signals termination of polymerization. Termination involves disassembly of the elongation machinery and the release of templates and products.

This is a general framework. We will not focus on every stage for each process, but rather on key stages that illustrate important concepts.

SLO 1. Explain how cells overcome three major challenges to replicate their genome.

To replicate its genome, the cell must overcome several challenges:

• There are two DNA strands to be replicated.
• The two strands run in opposite directions (they’re antiparallel).
• Replication must be accurate.
• Enormous amounts of DNA are replicated: 6 Gbp/cell (= 6×109 base pairs per cell).
• One and only one copy of each of the 46 chromosomes must be segregated into each daughter cell.

How is this done? Here we provide a cursory outline of DNA replication. Later in the course we will look more closely at replication, mitosis, and meiosis in the contexts of the cell division cycle, genetic inheritance, and cancer.

For now, there are three core concepts about DNA replication that you need to know:

• DNA r eplication is semiconservative (Fig. 1). This means that when a cell divides, the DNA duplexes in each daughter cell contain one of the parent cell’s original DNA strands (which is used as a template for polymerization, and one newly synthesized or nascent strand.

Discontinuous replication on one strand is necessary because the DNA polymerase can only add nucleotides to the 3´end of a nascent chain, but the template strands are antiparallel (Fig. 1).

An important difference between DNA and RNA polymerases is that DNA polymerases can only extend pre-existing nascent chains, while RNA polymerases can begin new chains from a single nucleotide. Thus, DNA polymerases invariably r equi r e a short RNA primer . The primer is made by a special RNA polymerase called primase (Fig. 2).

Each chromosome begins as one piece of double-stranded DNA. Replicating the very ends of the chromosomes, the telomeres , presents special problems on the lagging strand. Telomere DNA is therefore maintained by a special enzyme called telomerase.

SLO 2. Explain why different genes need to be transcribed at different rates, in different cells, at different times.

Most of the mammalian genome consists of non-coding DNA. A minority (~2%) of the human genome actually serves to encode mRNAs that are used as templates for protein synthesis. A similarly small fraction of the genome encodes other varieties of biologically important RNA molecules.

The fundamental question about gene expression is this: each of us has many different cell types, but only one genome. The different cell types are different because they make different proteins: muscle cells make contractile proteins; nerve cells have the enzymes needed to manufacture neurotransmitters; osteoblasts have the protein machinery needed to manufacture bone, and so on.

Moreover, even a single cell type needs to make different proteins at different times: we synthesize and secrete insulin (a peptide hormone) when we eat. We remodel entire tissues and organs throughout growth, in response to injury, and during pregnancy. How does thi s happen?

DNA — transcription —> RNA — translation —> polypeptide

The level of any given protein is a function of competing processes: synthesis and destruction. Protein synthesis requires an mRNA template, and the abundance of the mRNA encoding any given protein is also regulated by a balance of synthesis and destruction.

SLO 3. Describe the functions of the three mammalian RNA polymerases.

Rna transcription.

The cell makes RNA molecules that do different things:

• mRNA is the template for protein synthesis (translation).
• tRNA and rRNA are core parts of the protein synthesis machinery.
• Diverse RNA molecules are involved in regulating gene expression and other processes. Examples include micro RNA ( miRNA ) and long non-coding ( lncRNA ). Many other examples are emerging.

The various RNAs are made by three RNA polymerase (RNAP) enzymes:

• RNAP I makes most of the ribosomal rRNA — the most important part of the ribosome, the enzyme that synthesizes polypeptides.
• RNAP II makes all of the messenger mRNA — the templates for polypeptide synthesis.
• RNAP III makes transfer tRNA — the carrier of activated amino acids for polypeptide synthesis.

We will focus on transcription by RNAPII, because its activity controls the levels of mRNA templates for protein synthesis. The underlying principles by which RNAP I and III operate are similar.

SLO 4. Explain the structure of a mammalian gene, including regulatory and coding elements.

A gene contains two kinds of sequences:

• The transcription unit is the DNA sequence used as a template to synthesize RNA.
• Regulatory sequences tell RNAP where to initiate and terminate transcription. They allow cells to control which genes are actively transcribed (“expressed”), and which are silent. These sequences can be further sub-divided:
• The promoter sequence directs RNAP II and associated general transcription factors to the transcriptional start site .
• Enhancer sequences, usually 10-30 bp in length, bind transcription factors , or activator proteins , that instruct RNA polymerase to become active at the promoter. Each gene is controlled by different enhancer elements.
• Different cells contain specific sets of transcription factors . This is the main basis for cell-type-specific gene regulation! Enhancers can sit right next to the promoter, or tens of thousands of base pairs distant. Enhancers are usually upstream of the promoter but they can also be embedded within the transcription unit or even downstream of it.
• The terminator tells RNAP that it has reached the end of the transcription unit.

Sequence of Events in Transcription:

• Genomic DNA is packaged into chromatin . Transcription is regulated in part by how densely packaged a given gene is, and hence, how accessible its regulatory sequence elements are. Later, we will discuss how DNA packaging is controlled.
• To initiate transcription of a gene, RNAP II must be directed to the promoter. This is done by the General Transc r iption Factors (Figs. 5 and 6). The GTFs recognize and bind to promoter sequences. They place RNAP II at the start site. GTFs then locally melt the DNA at the promoter, separating the two DNA strands to form a transcription bubble .
• The GTFs are “general” transcription factors because they are always needed for initiation of transcription. However, the GTFs do not have the ability to regulate when RNAP actually initiates RNA synthesis.
• In other words, GTFs are necessary but not sufficient for initiation of transcription.
• Transcription factors that bind to regulatory promoter, so intervening DNA must loop out in order for transcription factors, coactivators, and the initiation complex (GTFs + RNAP II) to touch one another. enhancer sequences are needed to activate transcription by RNAP II and the GTFs (Figs. 5 and 6)
• Activating transcription factors “talk” to RNAP II and the GTFs by binding to coactivators that touch RNAP II and the GTFs (Fig. 6). Together, these events cause the pre-initiation complex — RNAP II and the GTFs — to initiate RNA polymerization. As we will see in a later session, transcription factors can also control chromatin structure.
• No primer is needed for RNA synthesis.
• NTP s are used for RNA synthesis, not 2-deoxy dNTP s.
• As RNAP II elongates the nascent mRNA chain, it moves along the template strand of the transcription unit (Fig. 5). The replication bubble moves as RNAP II “crawls” along the DNA template strand. In other words, the DNA double helix melts in front of RNAP II and re- hybridizes (anneals) behind it.
• When RNAP II reaches a terminator sequence (Fig. 5), the newly-synthesized RNA chain is released, RNAP is removed from the template strand, and the transcription bubble collapses.

The reason we care so much about the mechanics of RNAP II transcription is that this process controls which mRNA transcripts are produced, and in what abundance. This in turn controls the specific repertoire of proteins that can be made by each cell.

Fig. 7 shows the regulatory sequences of genes that encode some key proteins made only in specific kinds of cells: skeletal muscle cells, heart muscle cells, and cells in the lens of the eye.

Each type of DNA enhancer element sequence is recognized and bound by specific activating transcription factors — shown here by colored shapes. Humans have about 2,000 different transcription factors.

By producing specific combinations of transcription factors, each cell specifies which subsets of genes are actively transcribed, and in what quantities.

This concept i s so i mpor tant that it bears r epeat i ng:

The specific array of transcription factors, present in a given cell, shapes that cell’s pattern of gene expression — and thus, that cell’s overall protein complement, the cell’s identity (muscle, fibroblast, neuron, etc.) and its functional characteristics.

Another important point is that transcription factors are proteins. Consequently, the genes that encode tr anscri pt i on f act or s are t hemselves subject t o tr anscri pt ional r egulati on . By transcribing and translating specific transcription factors the cell can execute temporary or stable programs of gene expression in response to developmental cues and other signals, such as food or infection.

Fig. 8. Positive feedback loops maintain gene expression programs. Here, an “initiator” transcription factor binds an enhancer on the gene encoding a “terminal selector” transcription factor. When this gene is transcribed and the resulting mRNA is translated, the resulting protein binds to another enhancer in its own gene, and ensures that the terminal selector gene continues to be transcribed. The terminal selector also stimulates transcription of other genes needed for specific functions. Source: PNAS 110:7101

mRN A Pro cess ing and Ex port

DNA replication and RNA transcription both occur in the cell’s nucleus. However, proteins are synthesized in the cytoplasm. To serve as templates for protein synthesis, mRNA molecules must be exported from the nucleus to the cytoplasm. This occurs at a special portal in the nuclear membrane, the nuclear pore (Fig. 9). The nuclear pore is an immense molecular assemblage that precisely controls the passage of mRNA and other macromolecules into, and out of, the nucleus. This is another theme that we will encounter again and again: biosynthetic products made in one cellular organelle are shuttled to another location — as with stations on an assembly line.

In the nucleus, the initial “raw” RNA transcript made by RNAP II must be processed (Fig. 10). Only then is the mature mRNA exported from the nucleus to the cytoplasm, where it will be used as a template for protein synthesis.

• Later, in the cytoplasm, the 5´ cap will tell the protein synthesis machinery that the RNA bearing the cap is a messenger mRNA — a template for protein synthesis — and not some other type of RNA.
• The poly-A tail will signal export of the mRNA from nucleus to cytoplasm. It will also control the stability (the half-life) of the mRNA once it’s in the cytoplasm.
• The mRNA is spliced to remove introns and ligate (join) exons together. This step is somewhat involved and extremely important, so we’ll examine it in a bit more detail.

Differential mRNA splicing

The maturation of a large mRNA molecule may entail dozens of splicing reactions. In different cell types, these splicing reactions may be regulated so that not every exon ends up in each final, mature mRNA molecule. Consequently, a single t r anscription unit may encode more than one mRNA variant , with each derived from a different combination of exons (Fig. 11).

Differential splicing allows the ~20,000 protein-coding genes in the human genome to encode substantially more than 20,000 distinct mRNA templates and, thus, a much greater diversity of proteins.

To summarize:

• Transcription initiation controls how many mRNA transcripts get made.
• Different cells have different activating transcription factors.
• Different genes have different enhancers that bind different transcription factors.
• mRNA cap addition signals that the mRNA will be a template for protein synthesis.
• Differential splicing controls which exons are in the mature mRNA template, and thus the sequence of the resulting polypeptide.
• The poly-A tail (along with other features of the mRNA) controls nuclear export and the stability of the mRNA — how long it persists in the cytoplasm.

SLO 8. Outline the major steps of protein synthesis, including the roles of mRNA, tRNA, tRNA synthetases and ribosomes.

Here we summarize how polypeptide chains are synthesized and how they fold into their correct three-dimensional configurations.

The notes for this section begin with two charts: first, the assignments of RNA codons to amino acids (the genetic code); second, the chemical and physical properties of the 20 regular amino acids listed by features. This table also includes the rare amino acid selenocysteine, sometimes considered as the “21st amino acid,” although it is a modification of cysteine.

You do NOT need to memorize Tables 1 and 2 ! You do need to be able to apply their content.

Sour ce: W ikimedia

Protein Synthesis: the Genetic Code

Like nucleic acid synthesis, protein synthesis is a template-directed process. However, in protein synthesis, the process is a bit more complicated because the template does not directly interact with the polypeptide product. How this works has never been explained more plainly than by Francis Crick, in an astonishing proposal that he made in 1955:

…E a c h amino a c id would c ombin e c h e mi c all y, at a s p ec ial e n zy m e, with a s mall mol ec ul e whi c h , having a speci fi c hydr ogen- bonding sur f ace, could combi ne speci fi c all y with t he nucl eic acid t em plat e. This combination would al so suppl y t he ener gy necessary for polymerization… th e re would b e 20 different kind s of adaptor mole cu le , on e for e a ch amino acid , and 20 different enzymes to join t he amino acid to their adapt ors…

We now know that the “adapter” is tRNA . Crick’s proposal was correct in every detail save one: there are 61 possible combinations of 3-base mRNA codons (see Table 1), so there are more than 20 tRNA “adapters.” This system provides the biochemical basis of the genetic code — the rules through whi ch each 3-bas e codon in an m RNA templat e speci fi es one speci fi c amino aci d.

tRNA and aminoacyl tRNA synthetase enzymes

Each tRNA has 2 “business ends”:

• The anticodon pairs with the 3-base codon on the mRNA template. Look closely at the diagram (Fig. 13). Note that as in other nucleic acid hybrids, the two strands are antiparallel .
• The aminoacyl accept or si t e is a terminal adenosine (A) nucleotide, where the carboxyl group of the amino acid is esterified to the 3´-OH of the adenine. Note that this is a r elatively unstable, high – energy bond . It will make polypeptide elongation thermodynamically downhill, and hence favorable — exactly as predicted by Crick.
• Any given tRNA can (in principle) be esterified to any amino acid. However, the fidelity of tran sla tion d e p e nd s a b so lu te ly o n th e accurate ma t c h i n g o f a t RNA b ea r i n g a sp eci fi c anticodon to th e corresponding amino aci d .
• The job of coupling each amino acid to its corresponding tRNAs is done by 20 different enzymes: the aminoacyl tRNA synthetases (also called aaRS enzymes). This is an absolutely key point: the sp eci fi ci ty of th e geneti c cod e i s controlled by th e tRN A synth etases.

• In the first step, pyrophosphate (PP i ) is also released. As we saw in DNA and RNA polymerization, pyrophosphate is immediately destroyed by pyrophosphatase, making the first sub-reaction an irreversible committed step .
• Second, the aminoacyl group is transferred to the tRNA. The products are an aminoacyl tRNA, and AMP. Because we start with ATP, and end up with AMP and two inorganic phosphates, coupling of an amino acid to tRNA has an energetic cost of 2 ATP equivalents.

The Ribosom e

The “polypeptide polymerase” is the ribosome , an enormous ribonucleoprotein complex. The ribosome has two subunits . Each subunit contains both RNA and many different polypeptides.

• The small subunit (it is not small, just not as big as the large subunit!) is the “ decoding center .” The small subunit’s job is to match each codon on the template to a corresponding aminoacyl-tRNA. This is no easy task. There are 61 codons that specify 20 amino acids ( Table 1 ), so a large majority of the aminoacyl-tRNA molecules that enter the ribosome must be rejected.
• When a correct codon-anticodon interaction is detected by the small subunit, the large subunit catalyzes the peptidyltransfer reaction — the chemistry of polypeptide elongation.

Remarkably, although each ribosome subunit contains both peptides and rRNA, both the decoding center within the small subunit, and the peptidyltransfer center in the large subunit, are made of ribosomal rRNA . The enzymatic core of the r ibosome is a “r ibozyme ” . This is probably a relic of the ancient origin of ribosomes at the dawn of life, in the so-called RNA world.

• Initiation : the polymerase — the ribosome — must be placed precisely over the start codon on the mRNA template.
• Elongation : this is where template-mediated polymerization of the polypeptide occurs.
• Termination : A stop codon is identified, triggering release of the polypeptide and removal of the ribosome from the mRNA template.

Initiation of protein synthesis As with DNA replication and transcription, initiation of protein synthesis is pretty complicated. And since the frequency of initiation controls the rate of protein synthesis, this step is also highly regulated .

Regulation occurs at both a global level (the cell asks how much protein synthesis it can support overall, given the available energy and resources), and for specific mRNA transcripts, which are translated with different efficiencies.

Steps in translation initiation

• The small subunit and initiation factors crawl along the mRNA from 5´-to3´, scanning the mRNA for a start codon .
• In most cases the start codon is AUG . If you look at Table 1 , you’ll see that AUG encodes the amino acid methionine (Met, M). Thus, the first amino acid in a polypeptide is usually Met.
• Once the decoding center is accurately placed over the AUG start codon, the large subunit (in Fig. 15, the larger oval) docks onto the small subunit and mRNA, and the initiation factors fall off (dissociate). Now the elongation cycle can begin.

Two additional points about translation initiation must be emphasized.

First, as mentioned above, this step is highly regulated.   Initiation factors are largely responsible for this regulation.

Second, the accuracy with which the ribosome’s small subunit is placed over the start codon is critical : a positional error of ± 1 or 2 nucleotides will put the mRNA transcript out – of – frame , and result in a totally different, and incorrect, polypeptide sequence. Similarly, if a mutation in the genome inserts or deletes one or two DNA bases in the coding region of a gene, the  res ulting mRNAs will contain frameshift errors . When this happens, every codon following the frameshift will be incorrectly decoded during translation. Most often this also results in early termination of synthesis.

Note: There are two major, important differences in mRNA structure and translation initiation between eukaryotes (humans included) and bacteria.

• In bacteria, the first amino acid is u s ually a Met deriviative , fMet ( formylmethionine ). Pepti des beginning with f M et ar e recogni z ed by our innat e immune sys tem as a danger signal , b ec au se th e y c an indicate an acti ve bacterial infection. You will learn mor e about bacteria, danger signal s, and innat e immunity in the Infections and Immunity Block.

Polypeptide elongation

The assembled ribosome (large and small subunits) has three sites that can accommodate tRNA molecules: the A, P, and E sites. These names are shorthand for a minoacyl-tRNA, p eptidyl-tRNA, and e xit sites. It will become clearer in a moment why these names are used. The three sites are used in sequ ence. Here’s how it works (Fig. 17).

• At the A site, the ribosome samples incoming aminoacyl-tRNA (aa-tRNA) molecules. The r ibosome is l ooking for an aa – tRNA w ith a n a n tico d o n co rre ctly p airs w ith th e mRNA codon positioned under the A site . Dozens of incorrect aa-tRN A s are rejected for each correct match.
• The growing polypeptide chain, still esterified at its C (carboxyl)-terminus to a tRNA, resides in the P site.
• In the peptidyltransfer reaction , the nasent polypeptide is transferred from the peptidyl-tRNA sitting in the P site, to the aminoacyl residue sitting in the A site. This is counterintuitive. Inspect fig. 9 to see how it works.

A final point about energy. Two ATP equivalents used to charge each aa-tRNA. This powers the peptidyltransfer reaction. However, additional ATP equivalents are consumed during the tRNA selection and t ranslocation portions of the elongation cycle. In terms of both energy and materials, protein synthesis is very expensive. Many cell types use most of their energy on protein synthesis.

SLO9. Describe the basic ways in which microRNA (miRNA) molecules control gene expression.

MicroRNAs ( miRNAs ) are post-transcriptional regulators of gene expression. More than 2,000 miRNAs have been annotated in human genome. 60% of all human genes are estimated to be regulated by one or more miRNA. miRNAs are short noncoding RNAs , usually about 22 nucleotides long. miRNA molecules are formed through two major pathways :

• Many miRNA precursors are coded as stand-alone genes, which can be transcribed by RNA polymerase II. Note that in the figure above, the miRNA is derived from a Pol II transcript with a 5´cap and 3´poly-A tail.
• As they are not coding sequences, miRNA precursors can also be derived from intron sequences that are embedded within other mRNA precursor transcripts. In these cases, splicing excises the intron and its miRNA from the coding exons of the mature mRNA.

miRNA does not function alone. miRNAs bind to the Argonaute family of proteins in the cytoplasm. miRNA-Argonaute complexes bind to specific mRNA transcripts via complementary hybridization between the miRNA seed region, and target sequences in the 3’UTR of the targeted mRNA transcript. In most cases, miRNAs function to repress (decrease) the production of specific sets of proteins. miRNA-Argonaute-mRNA complex can repress protein expression in different ways:

• destabilization of the mRNA via shortening poly (A) tail;
• inhibition of translation initiation;
• cleavage and degradation of the target mRNA.

Note that these mechanisms are post-transcriptional , meaning that they operate on mature mRNA molecules in the cytoplasm. In contrast, transcription factors act in the nucleus to control the rate at which different mRNA molecules are synthesized (transcribed) by RNA polymerase II. The first miRNA was discovered in the nematode C. elegan s. Studies in humans have revealed that mutations in miRNAs can cause or contribute to various human diseases. For instance, mutation in miRNA-96 is linked to hereditary progressive hearing loss, and deletion of the miR-17~92 cluster causes skeletal abnormality and growth defects. Dysregulation of miRNA function is also implicated as a causative factor in several cancers.

Molecular Biology Copyright © by Alexey Merz; Timothy Cherry; and kullberm. All Rights Reserved.

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Transcription vs. Translation

Transcription is the synthesis of RNA from a DNA template where the code in the DNA is converted into a complementary RNA code. Translation is the synthesis of a protein from an mRNA template where the code in the mRNA is converted into an amino acid sequence in a protein.

Comparison chart

Localization.

In prokaryotes both transcription and translation occur in the cytoplasm due to the absence of nucleus. In eukaryote transcription occurs in the nucleus and translation occurs in ribosomes present on the rough endoplasmic membrane in the cytoplasm.

Transcription is performed by RNA polymerase and other associated proteins termed as transcription factors. It can be inducible as seen in the spatio-temporal regulation of developmental genes or consitutive as seen in case of house keeping genes like Gapdh.

Translation is performed by a multi-subunit structure called ribosome which consists of rRNA and proteins.

Transcription initiates with RNA polymerase binding to the promoter region in the DNA. The transcription factors and RNA polymerase binding to the promoter forms a transcription initiation complex. The promoter consists of a core region like the TATA box where the complex binds. It is in this stage that RNA polymerase unwinds the DNA.

Translation initiates with the formation of initiation complex. The ribosome subunit, three initiation factors (IF1, IF2 and IF3) and methionine carrying t-RNA bind the mRNA near the AUG start codon.

During transcription, the RNA polymerase after the initial abortive attempts traverses the template strand of DNA in 3’ to 5’ direction, producing a complementary RNA strand in 5’ to 3’ direction. As the RNA polymerase advances the DNA strand that has been transcribed rewinds to form a double helix.

During translation the incoming aminoacyl t-RNA binds to the codon (sequences of 3 nucleotides ) at A-site and a peptide bond is formed between the new amino acid and the growing chain. The peptide then moves one codon position to get ready for the next amino acid. The process hence proceeds in a 5’ to 3’ direction.

Termination

Transcription termination in prokaryotes can either be Rho-independent, where a GC rich hairpin loop is formed or Rho-dependent, where a protein factor Rho destabilizes the DNA-RNA interaction. In eukaryotes when a termination sequence is encountered the RNA nascent transcript is released and it is poly-adenylated.

In translation when the ribosome encounters one of the three stop codons it disassembles the ribosome and releases the polypeptide.

End Product

The end product of transcription is an RNA transcript which can form any of the following types of RNA: mRNA, tRNA, rRNA and non-coding RNA (like microRNA). Usually in prokaryotes the mRNA formed is polycistronic and in eukaryotes it is monocistronic.

The end product of translation is a polypeptide chain which folds and undergoes post translational modifications to form a functional protein.

Post Process Modification

During post transcriptional modification in eukaryotes, a 5’ cap, a 3’ poly tail is added and introns are spliced out. In prokaryotes this process is absent.

A number of post-translational modifications occur including phosphorylation, SUMOylation, disulfide bridges formation, farnesylation etc.

Antibiotics

Transcription is inhibited by rifampicin (antibacterial) and 8-Hydroxyquinoline (antifungal).

Translation is inhibited by anisomycin , cycloheximide , chloramphenicol, tetracyclin, streptomycin, erythromycin and puromycin .

Methods to measure and detect

For Transcription, RT-PCR, DNA microarray, In-situ hybridization, Northern blot, RNA-Seq is quite often used for measurement and detection. For Translation, western blotting, immunoblotting, enzyme assay, Protein sequencing, Metabolic labeling, proteomics is used for measurement and detection.

Crick’s central dogma: DNA ---> Transcription ---> RNA ---> Translation ---> Protein

Genetic code used during translation:

• wikipedia:Transcription (genetics)
• wikipedia:Translation (biology)
• Internet-Based Tools for Teaching Transcription and Translation - National Human Genome Research Institute
• Translation: DNA to mRNA to Protein - Nature

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12.15: Prokaryotic Translation

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Translation is similar in prokaryotes and eukaryotes. Here we will explore how translation occurs in E. coli , a representative prokaryote, and specify any differences between bacterial and eukaryotic translation.

The initiation of protein synthesis begins with the formation of an initiation complex. In E. coli , this complex involves the small 30S ribosome, the mRNA template, three initiation factors that help the ribosome assemble correctly, guanosine triphosphate (GTP) that acts as an energy source, and a special initiator tRNA carrying N -formyl-methionine (fMet-tRNAf Met ) (Figure 1). The initiator tRNA interacts with the start codon AUG of the mRNA and carries a formylated methionine (fMet). Because of its involvement in initiation, fMet is inserted at the beginning (N terminus) of every polypeptide chain synthesized by E. coli . In E. coli mRNA, a leader sequence upstream of the first AUG codon, called the Shine-Dalgarno sequence (also known as the ribosomal binding site AGGAGG), interacts through complementary base pairing with the rRNA molecules that compose the ribosome. This interaction anchors the 30S ribosomal subunit at the correct location on the mRNA template. At this point, the 50S ribosomal subunit then binds to the initiation complex, forming an intact ribosome.

In eukaryotes, initiation complex formation is similar, with the following differences:

• The initiator tRNA is a different specialized tRNA carrying methionine, called Met-tRNAi
• Instead of binding to the mRNA at the Shine-Dalgarno sequence, the eukaryotic initiation complex recognizes the 5′ cap of the eukaryotic mRNA, then tracks along the mRNA in the 5′ to 3′ direction until the AUG start codon is recognized. At this point, the 60S subunit binds to the complex of Met-tRNAi, mRNA, and the 40S subunit.

In prokaryotes and eukaryotes, the basics of elongation of translation are the same. In E. coli , the binding of the 50S ribosomal subunit to produce the intact ribosome forms three functionally important ribosomal sites: The A (aminoacyl) site binds incoming charged aminoacyl tRNAs. The P (peptidyl) site binds charged tRNAs carrying amino acids that have formed peptide bonds with the growing polypeptide chain but have not yet dissociated from their corresponding tRNA. The E (exit) site releases dissociated tRNAs so that they can be recharged with free amino acids. There is one notable exception to this assembly line of tRNAs: During initiation complex formation, bacterial fMet−tRNA fMet or eukaryotic Met-tRNAi enters the P site directly without first entering the A site, providing a free A site ready to accept the tRNA corresponding to the first codon after the AUG.

Elongation proceeds with single-codon movements of the ribosome each called a translocation event. During each translocation event, the charged tRNAs enter at the A site, then shift to the P site, and then finally to the E site for removal. Ribosomal movements, or steps, are induced by conformational changes that advance the ribosome by three bases in the 3′ direction. Peptide bonds form between the amino group of the amino acid attached to the A-site tRNA and the carboxyl group of the amino acid attached to the P-site tRNA. The formation of each peptide bond is catalyzed by peptidyl transferase , an RNA-based ribozyme that is integrated into the 50S ribosomal subunit. The amino acid bound to the P-site tRNA is also linked to the growing polypeptide chain. As the ribosome steps across the mRNA, the former P-site tRNA enters the E site, detaches from the amino acid, and is expelled. Several of the steps during elongation, including binding of a charged aminoacyl tRNA to the A site and translocation, require energy derived from GTP hydrolysis, which is catalyzed by specific elongation factors. Amazingly, the E. coli translation apparatus takes only 0.05 seconds to add each amino acid, meaning that a 200 amino-acid protein can be translated in just 10 seconds.

Termination

The termination of translation occurs when a nonsense codon (UAA, UAG, or UGA) is encountered for which there is no complementary tRNA. On aligning with the A site, these nonsense codons are recognized by release factors in prokaryotes and eukaryotes that result in the P-site amino acid detaching from its tRNA, releasing the newly made polypeptide. The small and large ribosomal subunits dissociate from the mRNA and from each other; they are recruited almost immediately into another translation init iation complex.

In summary, there are several key features that distinguish prokaryotic gene expression from that seen in eukaryotes. These are illustrated in Figure 2 and listed in Table 1.

Contributors and Attributions

• Biology. Provided by : OpenStax CNX. Located at : http://cnx.org/contents/[email protected] . License : CC BY: Attribution . License Terms : Download for free at http://cnx.org/contents/[email protected]

Gene to Protein

The majority of genes have the necessary instructions to produce the functional molecules known as proteins. Here, let’s learn the processes by which genes are converted to proteins.

Table of Contents

Replication, transcription, translation, frequently asked questions.

Genes are DNA sequences that control the synthesis of proteins and serve as bridges between phenotype and genotype. The amino acid sequences that make up proteins are specified by the protein-coding genes. Proteins, in turn, are in charge of directing almost all cellular processes. All cells maintain their genetic information through the three primary processes:

The base of biological inheritance is replication. The DNA of a cell is copied in this process. A single parental double-stranded DNA molecule is copied by the enzyme DNA polymerase into two daughter double-stranded DNA molecules. In subsequent steps, these DNA fragments are utilised to create RNA.

A single DNA strand serves as a template for the creation of a complementary strand of RNA during transcription.

• The attachment of the RNA polymerase enzyme to a DNA molecule is the initial step in transcription. The promoters, which are specialised sequences of 20 to 200 bases where various interactions take place, are the locations where binding takes place.
• The promoter DNA is bound by RNA polymerase and one or more general transcription factors. RNA polymerase synthesises new RNA nucleotides. These are complementary to the nucleotides present in one of the DNA strands.
• The freshly formed RNA strand is released when the RNA-DNA helix’s hydrogen bonds are disintegrated. The RNA might undergo additional processing like splicing, capping and polyadenylation.
• A messenger RNA (mRNA) molecule is thus created during the transcription of a gene.

Also Check: What Is Translation in Biology?

In the process of translation, mRNA or messenger RNA is decoded outside the nucleus in a ribosome to create a particular polypeptide or amino acid chain. The polypeptide chain undergoes further folding into secondary, tertiary and quaternary structures and carries out its specific tasks within the cell. This process happens in the following sequential steps:

• Initiation – The ribosome forms a protective shell around the target mRNA. Now, the first tRNA molecule is joined at the start codon.
• Elongation – The last accepted tRNA by the smaller subunit of ribosome transmits the amino acid it contains to the larger ribosomal subunit, which binds it to one of the previously admitted tRNAs. Following this, the ribosome translocates to the subsequent mRNA codon to complete the process and produce a polypeptide chain.
• Termination – The ribosome releases the polypeptide on reaching the stop codon. The next mRNA to be translated is taken up by the ribosomal complex, which is still intact.

Thus the genetic information is transferred from DNA(gene) to RNA and to Protein, which is a functional product. This is known as the central dogma.

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Introduction, mitochondrial transcription, maturation of the primary transcript, mitochondrial ribosome: structure and assembly, translation, concluding remarks, competing interests, author contribution, abbreviations, mitochondrial transcription and translation: overview.

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Caterina Garone , Michal Minczuk , Aaron R. D’Souza , Michal Minczuk; Mitochondrial transcription and translation: overview. Essays Biochem 20 July 2018; 62 (3): 309–320. doi: https://doi.org/10.1042/EBC20170102

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Mitochondria are the major source of ATP in the cell. Five multi-subunit complexes in the inner membrane of the organelle are involved in the oxidative phosphorylation required for ATP production. Thirteen subunits of these complexes are encoded by the mitochondrial genome often referred to as mtDNA. For this reason, the expression of mtDNA is vital for the assembly and functioning of the oxidative phosphorylation complexes. Defects of the mechanisms regulating mtDNA gene expression have been associated with deficiencies in assembly of these complexes, resulting in mitochondrial diseases. Recently, numerous factors involved in these processes have been identified and characterized leading to a deeper understanding of the mechanisms that underlie mitochondrial diseases.

Mitochondrial gene expression is central to maintaining cellular homoeostasis. The control of mitochondrial gene expression is unique in that its components have dual origins in the mitochondria (all RNAs) and nucleus (all protein factors). The regulation of the synthesis and degradation of mitochondrial (mt-) RNAs determines steady-state levels of mitochondrially encoded proteins allowing fine control of the mitochondrial energy metabolism. Therefore, the cell can adapt to changing environmental stresses and satisfy changing cellular energy demands. Defects in the mitochondrial gene expression can lead to respiratory chain dysfunction resulting in a multi-system disease phenotype, predominantly affecting muscular and neuronal tissues.

The mammalian mitochondrial genome is highly condensed as far as genetic information is concerned. The mitochondrial genome encodes 2 rRNAs, 22 tRNAs and mRNAs for 13 polypeptides of the oxidative phosphorylation (OxPhos) system. In some cases, the reduction of the mitochondrial genome has led to overlapping genes ( MT-ATP8/6, MT-ND4/4L and mt-tRNA Tyr /mt-tRNA Cys ). The entire mitochondrial genome is transcribed from both the strands as long polycistronic transcripts. These strands are named heavy (H) or light (L) based on their buoyancy in caesium chloride density gradients. The long polycistronic transcripts require multiple processing steps before individual RNA species become functional. After endonucleolytic cleavage of the primary transcript, the ribosomal RNAs undergo chemical modifications before it can function correctly within the mitoribosome, the tRNAs also undergo a large number of chemical modifications, in addition to further polymerization and aminoacylation, and the mRNAs are differentially polyadenylated. Finally, the mRNAs, tRNAs and the assembled mitoribosome come together in the translation apparatus where translational factors direct the progression of translation ( Figure 1 ).

Overview of human mitochondrial transcription, RNA processing and translation

The list of proteins mentioned in the figure are biased towards those that are associated with mitochondrial diseases, as explained in the article by Boczonadi et al. [ 1 ]. Aminoacyl-tRNA synthetases are abbreviated as xARS and xARS2.

In this article, we briefly overview the key stages of mitochondrial gene expression in humans, providing a useful basis for the article by Boczonadi et al. that deals with human diseases resulting from the defects in this pathway [ 1 ]. The main goal of our article is to present the basic mitochondrial function of protein factors that have been associated with mitochondrial disease, therefore, some proteins known to be involved in mitochondrial transcription and translation might have not been described in this brief overview.

Transcription of the mitochondrial genome originates in the major non-coding region containing the L-strand (LSP) and H-strand (HSP) promoters. The light strand promoter controls the transcription of eight of the tRNAs and the MT-ND6 gene. On the heavy strand, two H-strand two-promoter systems have historically been proposed, where HSP1 transcription produces a transcript containing tRNA Phe , tRNA Val and the two rRNAs (12S and 16S), while transcription from HSP2 generates a transcript that spans almost the entire genome [ 2–4 ]. This two-promoter model of H-strand transcription was proposed to explain the high abundance of the two rRNAs. However, more recent animal models [ 5 ] and in vitro [ 6 ] experiments suggest that heavy strand transcription is under the control of a single promoter and that the difference in rRNA abundance may be a consequence of differential turnover.

Transcription initiation

Transcription in human mitochondria is driven by a DNA-dependant RNA polymerase called POLRMT, which is structurally similar to RNA polymerases in T3 and T7 bacteriophages [ 7 , 8 ]. This includes high sequence homology to the C-terminal catalytic core of the enzyme [ 9 ]. At the N-terminal domain, POLRMT also contains two pentatricopeptide repeat (PPR) domains, commonly found in RNA-associated proteins, where they are required for site-specific interactions [ 7 , 10 ]. In contrast with bacteriophage polymerases, which can recognize promoter regions without auxiliary proteins, additional factors are required to perform this function by POLRMT. The initiation of transcription requires the association of POLRMT with mitochondrial transcription factor A (TFAM) and mitochondrial transcription factor B2 (TFB2M). TFAM is a DNA-binding protein, which, in addition to transcription activation, also packages DNA in the nucleoid [ 11 ]. TFB2M was produced as a result of a gene duplication event. TFB1M, the other product of this duplication event, is a ribosomal RNA methyltransferase (see below). Although TFB2M also contains a rRNA methyltransferase domain, the key function of this protein is DNA melting during the initiation of transcription [ 12–16 ]. Recent evidence shows that in the transcription initiation complex, at both the HSP and LSP, TFAM, bound to DNA, recruits POLRMT to the promoter via its N-terminal extension. TFB2M modifies the structure of POLRMT to induce opening of the promoter [ 14 , 17 , 18 ].

Transcription elongation

POLRMT requires an additional transcription elongation factor (TEFM) for the elongation stage [ 19 ]. Recombinant TEFM strongly promotes POLRMT processivity as it stimulates the formation of longer transcripts in vitro [ 20 ]. Also, depletion of TEFM in living cells leads to a reduction in promoter–distal transcription elongation products [ 19 ]. Transcription from LSP is often prematurely terminated around the conserved sequence block 2 (CSB2) of the major non-coding region of the mitochondrial genome. The short RNA molecule produced has been suggested to play a key role in priming DNA replication as multiple RNA to DNA transition sites clustering around CSB2 [ 21 , 22 ] (see also the article by Maria Falkenberg [ 23 ]). Stimulation of POLRMT processivity by TEFM prevents the formation of G-quadraplexes that inhibit the progression of the elongation complex at CSB2 [ 20 , 24 ]. The capability of TEFM to abolish premature transcription termination has been proposed to function as the switch from replication to the transcription of the LSP-derived primary transcript [ 24 ]. Recent structural work showed that TEFM contains a pseudonuclease core that forms a ‘sliding clamp’ around the mtDNA downstream of the transcribing POLRMT, interacting with POLRMT via its C-terminal domain [ 25 ].

Transcription termination

The mechanism of termination of HSP transcription is still unclear. It was previously suggested that mitochondrial termination factor 1 (MTERF 1 ) bends the mtDNA connecting the HSP1 promoter site and its apparent tRNA Leu(UUR) termination site. MTERF1 would then induce transcription termination through base flipping and DNA unwinding [ 26–28 ]. This model was originally proposed to explain the 50-fold higher abundance of mitochondrial rRNAs [ 27 ]. However, more recent evidence contradicts this hypothesis. Studies in MTERF1 knockout mice do not show an effect on rRNA steady-state levels [ 5 ]. Their increase in abundance is probably a product of increased stability rather than due to the presence of a different promoter. Moreover, it was also recently shown that transcription from the LSP is prematurely terminated by MTERF1 at the 3′-end of the mt-rRNA coding sequence. Binding of MTERF1 to this site prevents the replication fork from progressing into the mt-rRNA genes while they are being transcribed, whilst also preventing transcription of the antisense sequence of the rRNA [ 5 , 29 , 30 ].

Transcription from the heavy and light strand promoters produces long polycistronic transcripts. The mt-rRNA coding sequences and most of the protein coding sequences are separated by mt-tRNAs. Endonucleolytic excision of these mt-tRNAs releases the mRNAs and rRNAs – a concept known as the ‘tRNA Punctuation Model’ [ 31 , 32 ]. The processing of mt-tRNAs from the primary transcript is performed by RNase P and RNase Z at the 5′- and 3′-end respectively. Unlike previously characterized cytoplasmic and bacterial RNase P enzymes, which contain a catalytic RNA subunit, mammalian mitochondrial RNase P is an entirely proteinaceous heterotrimeric endonuclease. This enzyme is composed of a tRNA m 1 R9 methyltransferase, TRMT10C (MRPP1), a member of the short-chain dehydrogenase/reductase (SDR) family, SDR5C1 (HSD17B10, MRPP2), and a protein with homology to PiIT N-terminus (PIN) domain-like metallonucleases, PRORP (MRPP3) and cleaves the primary transcript at the 5′-end of tRNAs [ 33 ]. ELAC2 is an endonuclease that executes 3′-end maturation of both mitochondrial and nuclear pre-tRNAs [ 34–36 ].

The ‘tRNA punctuation model’ does not explain all the primary transcript cleavage events, as not all mRNAs are immediately flanked by tRNAs. Various Fas-activated serine/threonine kinase (FASTK) proteins have been shown to be required for mtRNA stability and the processing of precursors, especially the non-canonical cleavage sites. They all contain a conserved nuclease fold (RAP domain); however, endonucleolytic activity has not been shown for any of the FASTK proteins [ 37 ]. For example, depletion or knockout of FASTKD2 leads to the accumulation of various cleavage precursors, especially 16 rRNA and ND6 mRNA [ 38 , 39 ]. Additionally, FASTK has been implicated in MT-ND6 maturation and stability, and FASTKD5, similar to FASTKD4, regulates the maturation of those precursor RNAs that cannot be processed by RNase P and ELAC2 [ 39 , 40 ]. Furthermore, cross-linking immunoprecipitation (CLIP)-based analysis of FADTKD2 binding sites identified 16S rRNA and ND6 as its targets [ 38 ]. Recently, FASTKD4 was shown to be required for the stable expression of several mt-mRNAs, whereas FASTKD1 had the opposite effect on the stability of the MT-ND3 mt-mRNA. Interestingly, depletion of both FASTKD1 and FASTKD4 also caused a loss of MT-ND3, suggesting that the loss of FASTKD4 is epistatic [ 37 ]. Moreover, FASTKD4 has been suggested to be responsible for the cleavage of the MT-ND5-CYB precursor. A detailed characterization of how the FASTK proteins regulate the mitochondrial transcriptome is likely to be a subject of intense study in the near future.

mRNA maturation and stability

After excision from the primary transcript, all mRNAs, except MT-ND6, undergo 3′ polyadenylation. Polyadenylation in mitochondria is performed by a homodimeric polyadenylic acid RNA polymerase (mtPAP) [ 41–43 ]. Seven of thirteen mt-mRNAs do not contain a 3′ stop codon. In these cases, 3′ adenylation completes these stop codons and thus, the open reading frame. Polyadenylation of bacterial transcripts generally mark them for degradation, whereas addition of poly(A) tails to eukaryotic, nuclear-encoded mRNA is necessary for their stability. However, in mammalian mitochondria the effect of polyadenylation on steady-state levels is mRNA-specific. For example, deadenylation decreases complex IV mt-mRNA and increases complex I mt-mRNA levels [ 41 , 44–46 ].

The stability of HSP-derived mitochondrial transcripts is regulated by leucine-rich penticopeptide rich domain containing protein (LRPPRC) [ 47 ]. Loss of LRPPRC reduces the steady-state levels of mRNAs whilst not affecting rRNAs and tRNAs, consequently leading to a translation defect and loss of respiratory complexes [ 48–51 ]. The presence or absence of LRPPRC in the mitochondria correlates with the level of mt-mRNA polyadenylation [ 52 , 53 ]. As such, LRPPRC mouse knockout models display a loss in HSP-derived transcripts, loss of poly(A) tails and a severe translational defect [ 50 ]. More recent data show that LRPPRC is a mt-mRNA chaperone that relaxes secondary structures, therefore, facilitating RNA polyadenylation and coordinated mitochondrial translation [ 54 ]. Following translocation into mitochondria, LRPPRC forms a complex with a stem–loop interacting RNA-binding protein (SLIRP) [ 55 ]. Within this complex, SLIRP stabilizes LRPPRC by protecting it from degradation [ 56 ], whilst being dispensable for polyadenylation of mtDNA-encoded mRNAs [ 56 ]. The LRPPRC–SLIRP complex has also been shown to suppress their degradation of mt-mRNAs [ 57 ].

Human mitochondrial RNA decay is mediated by a complex of polynucleotide phosphorylase (PNPase) and human Suv3 protein (hSuv3) [ 58 ]. PNPase is a 3′–5′ exoribonuclease which has been shown to localize to the intermembrane space [ 59 ], and also in distinct foci with hSuv3p and mitochondrial RNA [ 58 ]. Knockdown of PNPase leads to the increase in the half-life of mitochondrial transcripts and the accumulation of RNA decay intermediates [ 58 ]. hSuv3p is an NTP-dependent helicase. The lack of a functioning hSuv3 helicase leads to the accumulation of aberrant RNA species, polyadenylated molecules and degradation intermediates [ 60 ]. Recent evidence shows that exposure to the intercalating agent ethidium bromide (EtBr), which disrupts tRNA secondary structure, causes them to be polyadenylated. Subsequent withdrawal of EtBr causes the polyadenylated tRNAs to be rapidly degraded by the PNPase–hSuv3 degradosome [ 61 ]. Knockdown of PNPase leads to lengthening of the poly(A) tails due to inhibited tRNA turnover [ 61 ]. Controversially, the localization of PNPase in the intermembrane space has led to implications that it plays a role in the import of endogenous RNA into mitochondria. However, various pieces of evidence suggest that this is not the case and that it primarily functions in the RNA degradosome [ 62 ].

tRNA maturation

The mt-tRNAs undergo extensive post-transcriptional maturation including chemical nucleotide modifications and CCA addition at the 3′-end deadenylation. One of the key tRNA positions of chemical modification is the ‘wobble’ base (position 34) at the anticodon of the tRNAs. During translation, the appropriate amino acyl-tRNA is positioned in the mitoribosome through the accurate recognition of a cognate mRNA codon. However, since, many codons code for the same amino acid, the first position of the tRNA anticodon is chemically modified to facilitate non-Watson–Crick base pairing, therefore expanding codon recognition during mitochondrial translation. Some of the enzymes involved in modifying this position include: NSUN3 and ABH1 which are required for the introduction of 5-formylcytosine at the wobble position (f 5 C34) of mt-tRNA Met [ 63–66 ], MTO1 and GTPBP3 are required for the biogenesis of 5-taurinomethyluridine (τm 5 U) [ 67 , 68 ], and MTU1 (TRMU) which catalyses the 2-thiolation of 5-taurinomethylridine to form τm 5 s 2 U of a subset of mt-tRNAs [ 69 , 70 ].

In addition to the modification of the wobble position, position 37 downstream of the anticodon is also frequently chemically modified in order to facilitate stable codon–anticodon interactions and, therefore, increasing accuracy and fidelity of mitochondrial translation. Examples of enzymes that are responsible for modifying mt-tRNA position 37 include TRIT1 responsible for the introduction of an isopentenyl group onto N 6 of 37 adenine (i 6 A37) in a small subset of mt-tRNAs [ 71 ] or TRMT5 which introduces N 1 -methylation of the 37 guanosine (m 1 G37) [ 72 ].

Pseudouridine (Psi), the most common RNA modification, is often referred to as the fifth nucleotide. It is a structural isomer of uridine produced by a rotation around the N3–C6 axis. Psi is generally associated with a stabilizing role, by providing structural rigidity to RNA molecules regardless of sequence or structure, and has been detected in several mt-tRNAs. PUS1 is a pseudouridine synthetase which modifies U27 and U28 on mt-tRNAs [ 73 , 74 ]. Recently, a pseudouridine synthetase, RPUSD4, was characterized as introducing pseudouridine at position 39 of tRNA Phe [ 75 ]. Other putative PUSs have been identified as necessary for mitochondrial translation, including RPUSD3 and TRUB2; however, their exact mtRNA targets remain to be further characterized [ 76 , 77 ].

The CCA found at the 3′-end of all mature mt-tRNAs is not encoded by mtDNA and is instead post-transcriptionally synthesized by tRNA-nucleotidyltransferase 1 (TRNT1): TRNT1 does not require a template sequence, instead preferentially selecting CTP and ATP for polymerization [ 78 ]. Similarly, a non-encoded 5′ guanine on mt-tRNA His is post-transcriptionally added by 3′–5′ polymerase activity probably provided by THG1L [ 79 ]. The 3′ ends of several mt-tRNAs undergo spurious, mistargeted adenylation precluding correct aminoacylation at the 3′-end (see below). A 3′–5′ exonuclease, PDE12, is required for the removal of these spurious poly(A) tails [ 45 , 80 ].

tRNA aminoacylation

Mitochondrial translation requires that each tRNA is charged with the cognate amino acid. This process is mediated by the mitochondrial aminoacyl-tRNA synthetases (ARS2s), which are encoded by nuclear genes. Of these, 17 ARS2s are unique to the mitochondria, while GARS (Glycyl-tRNA synthetase) and KARS (Lysyl-tRNA synthetase) are encoded by the same loci as the cytoplasmic enzymes, with the mitochondrial isoforms being generated by alternative translation initiation (GARS) [ 81 ] or alternative splicing (KARS) [ 82 ]. Interestingly, glutaminyl mt-tRNA (mt-tRNA Gln ) is aminoacylated by an indirect pathway, in which it is first charged with glutamic acid (Glu) by mitochondrial glutamyl-tRNA synthetase (EARS2), after which the Glu-mt-tRNA Gln is transamidated into Gln-mt-tRNA Gln , using free glutamine as an amide donor [ 83 ]. This latter conversion is performed by GatCAB, the glutamyl-tRNA Gln amidotransferase protein complex, which consists of three subunits: GatA (QRSL1), GatB (GATB) and GatC (GATC) [ 84 ].

The mitoribosome consists of a large 39S subunit (mtLSU) and a small 28S (mtSSU) subunit. Compared with the bacterial ribosome, the mammalian mitoribosome has reduced rRNA components. To compensate for this, 36 mitochondria-specific proteins have been recruited to the ribosome, primarily found at the periphery of the complex surrounding a highly conserved catalytic core [ 85–89 ]. In the bacterial ribosome, a 5S rRNA acts as a scaffold interconnecting the LSU, SSU and the tRNAs in the intersubunit space. However, recent structures of the mitoribosome instead identified the recruitment of a mitochondrially encoded tRNA to this site (tRNA Val in humans and rat, tRNA Phe in porcine and bovine mitochondria) [ 88 , 90 , 91 ].

The maturation of the mitoribosome requires the post-transcriptional processing of the catalytic rRNA in addition to the import and assembly of about 80 nuclear-encoded proteins (MRPL and MRPS proteins). As for other ribosomes, both the small subunit and the large subunit rRNA undergo chemical nucleotide modifications. These modifications include base methylations, 2′- O -ribose methylations and pseudouridylation, with several enzymes responsible for these modifications having been identified (TRMT61B [ 92 ]; TFB1M [ 93 ]; NSUN4 [ 94 ]; MRMs [ 95 ]; RPUSD4 [ 76 ]; reviewed in [ 96 ]). For example, the A-loop region of the 16S rRNA is methylated at position U1369 and G1370 (human mtDNA numbering). This site directly interacts with the aminoacyl-tRNA [ 88 , 97 ]. U1369 is methylated by MRM2, which has been shown to interact with the mtLSU. Depletion of the protein leads to defective biogenesis of the mtLSU and consequently, a deficiency in translation [ 97 ]. Also, several protein factors not directly involved in rRNA modification have been identified to coordinate the assembly of the mitoribosome reviewed in [ 96 ]. For example, ERAL1, a homolog of the bacterial Era protein that belongs to the conserved family of GTP-binding proteins, has been proposed to act as an RNA chaperone that stabilizes 12S mt-rRNA during mtSSU assembly [ 98 ].

Many of the proteins involved in mitoribosome assembly and the post-transcriptional processing of the nascent transcript, including FASTK proteins, ELAC2 or RNase P (see above) are found in distinct foci called mitochondrial RNA granules (MRG). This compartmentalization has been proposed to facilitate more efficient and accurate gene expression [ 40 , 99 , 100 ]. It has been suggested that an integral inner membrane protein, RMND1, stabilizes and anchors the mitochondrial ribosome at the inner membrane, adjacent to MRGs where the mRNAs are produced and processed [ 101 ]. However, the exact function and mechanism of this protein is still unclear.

Mitochondrial translation is fully dependent on various nuclear-encoded regulatory proteins. In the mammalian mitochondria, the mitochondrial initiation factors, mtIF2 and mtIF3, control the initiation of translation [ 102 ]. During initiation, mtIF3 positions the AUG or AUA initiation codons of the mRNA at the peptidyl (P) site in the mtSSU and prevents the premature association of the mtLSU and mtSSU [ 103–105 ]. As in all protein synthesis systems, translation in mitochondria is initiated with a methionine residue. However, mitochondria differ in that only a single tRNA Met is used for both initiation and elongation. Discrimination, instead, is achieved through a post-transcriptional modification, with the aminoacylated initiator mt-tRNA Met being subjected to formylation of methionine (fMet), thereby increasing its affinity for mtIF2 [ 106 ]. mtIF2 directs the association of the fMet-tRNA Met with the mRNA, and guides the assembly of the mitochondrial monosome and the initiation of translation [ 107 , 108 ].

Translation in mammalian mitochondria differs from that of the cytoplasm or that of the yeast mitochondria in part due to the general absence of 5′-untranslated regions (UTRs) on mRNAs, gene-specific RNA cis -acting regulatory elements and introns. In yeast, a 5′-UTRs allow mRNA-specific, translational activators to bind and direct the mRNA into the mitoribosome for translation. However, in mammalian mitochondria, such mRNA regulatory elements have not been identified. Hence, alternative mechanisms are in place for the regulation of translation. Unlike UTR-based regulation, these protein factors have to bind directly to the mitochondrial transcript and affect gene expression. For example, various protein factors such as TACO1, MITRAC or C12orf62 have been recruited to modulate the translation of complex IV subunit CO1. Absence of any of these protein leads to a complex IV deficiency [ 109–111 ].

Elongation of translation is mediated by mitochondrial elongation factors, EFTu (TUFM), EFTs (TSFM) and EFGM (GFM1) [ 112 , 113 ]. In elongation, EFTu forms a complex with GTP and aminoacyl tRNA. It directs the tRNA to the acceptor (A) site where the tRNA base pairs with the mRNA at the codon–anticodon site. The hydrolysis of GTP catalyses peptide bond formation. EFTu is released and the GTP:EFTu complex is re-established by EFTs [ 114 ]. EFG1-mt causes the release of the deacetylated tRNA from the P-site, translocates the peptidyl-tRNAs from the A and P site to the P and exit (E) site, also causing the mRNA to move along by one codon.

Termination of mitochondrial translation is finally triggered by the presence of a stop codon at the A-site. Four mitochondrial proteins with homology to ribosome release factors have been identified in humans, including mtRF1, mtRF1a, C12orf65 and ICT1. These factors are characterized by the presence of a tripeptide GGQ motif that confers peptidyl-tRNA hydrolase activity [ 115 , 116 ]. Structural analysis of mtRF1 suggested that it is capable of recognizing the UAA and UAG stop codons, targeting ribosomes with a vacant A-site [ 117 , 118 ]. However, it does not exhibit release activity in vitro [ 119 ]. mtRF1a catalyses the hydrolysis of peptidyl tRNA at the UAA and UAG stop codons [ 120 ]. mtRF1a has been proposed to be sufficient for the termination of translation of all 13 mtDNA-encoded polypeptides, despite the mRNAs for MT-CO1 and MT-ND6 lacking the UAA and UAG stop codons at the end of the open reading frame (ORF). Instead, these ORFs contain in-frame AGA and AGG as the last codons respectively. AGA and AGG are used to encode Arg according to the universal genetic code; however, they are not used for this purpose in any of the mitochondria ORFs. Fine mapping of the termination codons of the mRNAs showed that these two mRNAs terminate at the UAG stop codon possibly created as a result of a −1 frameshift of the mitoribosome [ 121 , 122 ]. Initially, ICT1 was suggested as the protein that performed the termination function at the AGA and AGG codons. However, neither ICT1 nor C12orf65 release factor homologues containing the specific domains required for UAA and UAG stop codon recognition [ 116 ]. Recent evidence also suggests that ICT1 is capable of inducing hydrolysis of the peptidyl tRNAs in stalled mitoribosomes [ 119 , 123 ]. Since ICT1 is incapable of performing the peptidyl hydrolase activity, where the RNA template extends 14 nucleotides beyond the A-site, as is the case in MT-CO1 and MT-ND6, ICT1 may not be directly involved in the termination of translation of these two mRNAs [ 124 , 125 ].

Finally, after the release of the polypeptide, mitochondrial ribosomal recycling factors, mtRRF and EFG2 (also known as RRF2M, a homologue of EFGM) catalyse the release of the mRNAs, deacetylated tRNAs and the ribosomal subunits [ 126 , 127 ].

Diseases affecting mitochondrial transcription and translation, as described in the article by Boczonadi et al. [ 1 ], can have multi-systemic and severe manifestation. The development of novel, treatments of these mitochondrial diseases can be made more effective through a deeper understanding of the underlying mechanisms that cause them.

Progression of mitochondrial transcription and translation requires the sequential recruitment of different, nuclear-encoded initiation, elongation and termination factors.

Almost the entire mitochondrial genome is transcribed as long polycistronic transcripts.

Maturation of the transcripts requires endonucleolytic cleavage, but not all mRNAs are produced through RNase P and RNase Z function.

Mitochondrial mRNA steady-state levels are mainly controlled post-transcriptionally.

The role mitochondrial mRNA polyadenylation is not fully understood.

Mitochondrial tRNAs undergo extensive chemical modifications, including the addition and removal of nucleotides during their maturation.

Aminoacyl tRNA-synthetases charge tRNAs with their cognate amino acid, many of which are unique to the mitochondria.

Mammalian mitoribosomes differ considerably from other ribosomes as far as architecture and composition are concerned, with the key differences being the reversed protein:RNA mass ratio, incorporation of mtDNA-encoded structural tRNA and many novel, mitochondria-specific protein components.

The assembly of the mitoribosome assembly pathway is likely to be considerably different from its bacterial counterpart, implying the presence of mitochondria-specific regulatory factors.

We thank Dr Christopher Powell and other members of the Mitochondrial Genetics group at the MRC MBU, University of Cambridge for stimulating discussion during the course of this work.

The authors declare that there are no competing interests associated with the manuscript.

This work was supported by the Medical Research Council, U.K. [MC_U105697135 and MC_UU_00015/4].

A.R.D. contributed to drafting this review. A.R.D. and M.M. contributed to revising it and approved the final version. A.R.D. prepared the figure.

aminoacyl-tRNA synthetase

conserved sequence block 2

Fas-activated serine/threonine kinase

H-strand promoter

human Suv3 protein

leucine-rich penticopeptide rich domain containing protein

L-strand promoter

mitochondrial termination factor 1

polynucleotide phosphorylase

stem–loop interacting RNA-binding protein

tRNA-nucleotidyltransferase 1

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Study the following questions to prepare for your exam. The answers provided by your classmates in class can be viewed by clicking on the "Answer" link.

1) Living organisms use DNA as their genetic material. Outline how DNA is replicated within the cells of living organisms. [8] ..... ( Answer )

2) Outline what occurs during transcription. [10] ..... ( Answer )

3) Outline what occurs during translation. [9] ..... ( Answer )

4) Explain how complementary base pairing is used in replication, transcription and translation.  [10] ..... ( Answer )

5) Draw and label a simple diagram to show how DNA is constructed from sugars, phosphates and bases [6] ..... ( Answer )

6) Outline the structure of a DNA nucleotide. [5] ..... ( Answer )

7)  Outline the structure of a RNA nucleotide [5] ..... ( Answer )

8) List three differences between the structure of DNA and RNA. [3] ..... ( Answer )

9) Define the term degenerate as it applies to the genetic code. [1] ..... ( Answer )

10) Define the term universal as it applies to the genetic code. [1] ..... ( Answer )

11) Outline differences between eukaryotic and prokaryotic chromosomes. [8] ..... ( Answer )

12) Explain the one gene, one polypeptide rule. [1] ..... ( Answer )

13) Define the purpose of each of the three types of RNA. [3] ..... ( Answer )

essay - transcription, translation and pronunciation online

Transcription and pronunciation of the word " essay " in British and American variants. Detailed translation and examples.