What Role Does Transcription Play In Protein Synthesis – Home » Student Resources » Chemistry Textbooks Online » CH450 and CH451: Biochemistry – Defining Life at the Molecular Level » Chapter 11: Translation
Figure 11.31 One stretching cycle. (Left) During one round of amino acid elongation on the nascent peptide, the EF-Tu protein binds to a cognate aa-tRNA molecule and transports it to the A-site of the ribosome. Hydrolysis of GTP by EF-Tu leads to hybridization of the tRNA anticodon with the mRNA codon and causes EF-Tu (bound to GDP) to dissociate from the ribosome. (Center) After EF-Tu dissociation, a peptide bond is formed leading to transfer of the nascent peptide from the P-site tRNA to the A-site tRNA. (Right) Peptide bond formation leads to a conformational change in the ribosome that allows binding of EF-G (GTP Bound) near the A-site of the ribosome. Rapid hydrolysis of GTP by EF-G causes a large conformational shift in the protein that twists the ribosomal large subunit and moves the bound tRNA from the A- to the P-site; from P- to E-site; or from the E-site to exit from the ribosome.
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What Role Does Transcription Play In Protein Synthesis
EF-G is a GTP hydrolase protein that binds to the A-site of the ribosome. The EF-G protein has a high flexibility that allows it to act as a hinge. EF-G folding depends on GTP hydrolysis. Thus, when bound to the ribosome, the rapid hydrolysis of GTP acts as a powerful shock that folds the EF-G protein and causes a conformational change in the ribosome that allows the translocation of tRNA and mRNA residues. Translocation of tRNA is accompanied by large collective movements of the ribosome: relative rotation of ribosomal subunits and movement of the L1-stalk (Fig. 11.32). The L1 stem, which is the flexible part of the large subunit, contacts and moves together with the tRNA from the P to the E site. Once in the EF-G-GDP form, the factor rapidly dissociates from the ribosome, opening the A-site for the recruitment of the next aa-tRNA molecule. The elongation cycle will continue to repeat itself until a termination codon is reached.
Messenger Rna (mrna) — Overview & Role In Translation
Figure 11.32 Movement of the large subunit of the ribosome during translocation. (a) Pre-translocation structure of a ribosome with tRNA at A and P sites (green, brown). The L1 stem of the large subunit is shown in purple. (b) Movements accompanying tRNA translocation.
The elongation phase of eukaryotic translation is very similar to prokaryotic elongation. Essentially, mRNA is decoded by the ribosome in a process that requires the selection of each RNA for aminoacyl-transfer (aa-tRNA), which is dictated by the codon of the mRNA at the ribosomal acceptor site (A), the formation of a peptide bond, and the movement of both tRNA and mRNA through the ribosome (Fig. 11.33) A new amino acid is incorporated into a nascent peptide at a rate of approximately one every six seconds. The first step of this process requires guanosine triphosphate (GTP)-bound eukaryotic elongation factor 1A (eEF1α) to recruit the aa-tRNA to the aminoacyl (A) site, which has an anticodon loop cognate to the codon sequence of the mRNA. The anticodon of this sampling tRNA does not initially base pair with the A-site codon. Instead, the tRNA is dynamically remodeled to form a codon-anticodon helix, which stabilizes the binding of the tRNA-eEF1α-GTP complex to the ribosomal A site. This helical structure is energetically favorable for cognate or correct pairing, and thus distinguishes between non-cognate or unpaired and individual mismatched or near-cognate species. This is important for decoding accuracy because it provides a mechanism for rejecting non-cognate tRNA carrying the wrong amino acid. tRNA-codon pairing induces GTP hydrolysis by eEF1α, which is then ejected from the A site. In parallel with this process, the ribose undergoes a conformational change that stimulates contact between the 3′ end of the aa-tRNA at the A site and the tRNA carrying the polypeptide chain at the peptidyl (P) site. The repositioning of the two tRNAs [A to the P site and P to the exit (E) site] leads to the formation of a ribosome-catalyzed peptide bond and transfer of the polypeptide to aa-tRNA, thereby extending the polypeptide by one amino acid. The second phase of the elongation cycle requires a GTPase, eukaryotic elongation factor 2 (eEF2), which enters the A-site and, by hydrolysis of GTP, causes a conformational change of the ribosome. This stimulates translocation of the ribosome to allow the next aa-tRNA to enter the A-site, thus starting a new cycle of elongation.
Figure 11.33 Elongation phase of eukaryotic translation. This scheme represents the four basic steps of eukaryotic translation elongation. The ribosome contains three tRNA binding sites: aminoacyl (A), peptidyl (P), and exit (E) sites. In the first step of peptide elongation, tRNA, which is complexed with eIF1 and GTP and contains the cognate anticodon of the mRNA coding sequence, enters the A site. Recognition of the tRNA leads to GTP hydrolysis and the expulsion of eEF1 from the A site. In parallel, the deacylated tRNA at the E site is ejected. The A site and the tRNA site P interact, allowing ribosome-catalyzed peptide bond formation. This involves transfer of the polypeptide to the aa-tRNA, thereby extending the resulting polypeptide by one amino acid. eIF5A allosterically assists in the formation of certain peptide bonds, e.g. proline-proline. eEF2 then enters the A site and, through GTP hydrolysis, induces a conformational change of the ribosome and stimulates translocation. The ribosome is then in the correct conformation to accept the next aa-tRNA and begin a second cycle of elongation.
Termination of bacterial protein synthesis occurs when a stop codon is presented at the ribosomal A site and is recognized by a class I release factor, RF1 or RF2. These release factors (RFs) have different but overlapping specificities, with RF1 reading UAA and UAG and RF2 reading UAA and UGA, with strong sense codon discrimination. RFs are multidomain proteins, where binding and stop codon recognition by domain 2 at the decoding site causes the universally conserved GGK motif of domain 3 to insert into the A-site of the PTC, some 80 A away from the decoding site. This event triggers hydrolysis of the peptidyl-tRNA bond at the P-site of the PTC, and the resulting peptide chain can then be released through the ribosomal exit tunnel (Figure 11.34). After peptide release, RF1 and RF2 dissociate from the post-termination complex. Dissociation is accelerated by a class II release factor called RF3, which functions as a translational GTPase that binds and hydrolyzes GTP during termination.
Intro To Gene Expression (central Dogma) (article)
While RF3 increases the efficiency of peptide hydrolysis, it is not an essential protein for the process. In gene knockout studies, RF3 is essential for Escherichia coli growth, and its expression is not conserved across bacterial lineages. For example, RF3 is absent in the thermophilic model organisms of the genera Thermus and Thermatoga and in the infectious Chlamidiales and Spirochaetae. This means that both RF1 and RF2 are able to complete the termination cycle independently of RF3 or that other GTPases from the elongation or translation initiation phase can compensate for the action of RF3.
The release factors RF1 and RF2 adopt an open conformation (Figure 11.34) on the 70S ribosome, which is clearly different from the closed conformation observed in the crystal structures of free RFs. The conformational equilibrium of free RFs in solution shows that this open conformation dominates with about 80%.
Figure 11.34. Bacterial 70S ribosome termination complex with RF2. (A) View of the ribosome termination complex with E and P-site tRNAs (brown), mRNA (green), and RF2 (dark blue). (B) Close-up view of the hinge region of RF2 between domains 1 and 4 used for virtual screening, where the putative binding region is marked by the docked ligand (red).
During peptide hydrolysis, RF factors induce rotational and conformational changes within the ribosome that allow the binding of ribosome recycling factors (RRF) and EF-G GTPase, leading to dissociation of the large subunit from the small subunit and release of mRNA (Figure 11.35).
Transcription And Splicing: A Two‐way Street
Figure 11.35 Termination of translation. When the stop codon enters the A-site of the ribosome, RF1 or RF2 enters the A-site and binds to the mRNA. This leads to protein hydrolysis and release through the exit tunnel. Binding of RF3 and GTP hydrolysis causes dissociation of the RF factor and a conformational change in the structure of the ribosome. Subsequent binding of ribosome recycling factors, RRF and EF-G causes dissociation of the large and small ribosomal subunits and release of mRNA.
In eukaryotes and archaea, on the other hand, one omnipotent RF reads all three stop codons. Although the mechanism of translation termination is essentially the same, there is neither sequence nor structural homology between bacterial RF and eukaryotic eRF1, except for the universally conserved GGK motif that is required for peptide hydrolysis from tRNA. eRF3
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