What Are Two Steps Of Protein Synthesis – Home » Student Resources » Online Chemistry Textbooks » CH450 and CH451: Biochemistry – Determining Life at the Molecular Level » Chapter 11: Interpretation

Figure 11.31 An Elongation Cycle. (Left) During one round of amino acid elongation of a nascent peptide, the EF-Tu protein binds to the cognate aa-tRNA molecule and delivers it to the A-site of the ribosome. GTP hydrolysis by EF-Tu leads to the hybridization of the anticodon of tRNA with the codon of mRNA and causes the dissoication of EF-Tu (GDP-bound) from the ribosome. (Center) After EF-Tu dissociation, a peptide bond is formed leading to the transfer of the nascent peptide from the tRNA at the P-site, to the tRNA at the A-site. (Right) Peptide bond formation leads to a conformational change in the ribosome that allows the 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 large subunit of the ribosome and moves bound tRNAs from the A- to the P-site; from P- to E-site; or from the E-site to the exit of the ribosome.

What Are Two Steps Of Protein Synthesis

EF-G is a GTP hydrolase protein that binds to the A-site of the ribosome. The EF-G protein has high flexibility that enables it to act as a hinge. EF-G folding depends on GTP hydrolysis. Thus, when binding to the ribosome, the rapid hydrolysis of GTP acts as a power stroke that folds the EF-G protein and causes a change in the conformation of the ribosome that is able to translate the tRNA residues and the mRNA. The translocation of tRNAs is accompanied by large collective movements of the ribosome: relative rotation of the ribosomal subunits and L1-stalk motion (Fig. 11.32). The L1 stalk, which is a flexible part of the large subunit, is in contact and moves with the tRNA from the P to the E site. Once in the EF-G-GDP form, the factor quickly dissociates from the ribosome, opening the A-site for the recruitment of the next aa-tRNA molecule. The elongation cycle will continue to repeat until the termination codon is reached.

Translation In Protein Synthesis

Figure 11 .32 Large Movements of the Large Subunit of the Ribosome During Translocation. (a) Pre-translocation ribosome structure with tRNAs in A and P sites (green, brown). The L1 stalk 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. In essence, the mRNA is decoded by the ribosome in a process that requires the selection of each aminoacyl-transfer RNA (aa-tRNA), which is dictated by the mRNA codon of the ribosome acceptor (A) site, peptide bond formation and movement of both. tRNAs and the mRNA by the ribosome (Fig. 11.33) A new amino acid is incorporated into a nascent peptide at a rate of approximately one every sixth of a second. The first step in this process requires guanosine triphosphate (GTP)-bound eukaryotic elongation factor 1A (eEF1α) to recruit aa-tRNA to the aminoacyl (A) site, which has an anticodon loop adjacent 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 dynamically changes to create a codon-anticodon helix, which stabilizes the binding of the tRNA-eEF1α-GTP complex to the ribosome A site. This helical structure is energetically favorable for cognate or exact matches, and therefore discriminates between non-cognate or unpaired and single mismatched or near-cognate species. This is important for decoding accuracy because it provides a mechanism to reject a non-cognate tRNA that carries an inappropriate amino acid. Pairing of tRNA and codon induces GTP hydrolysis by eEF1α, which is displaced from the A site. In parallel with this process, the ribosome 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 transfer of the position of the two tRNAs [A to the P site and P to the exit (E) site] results in ribosome-catalysed peptide bond formation and the transfer of the polypeptide to the aa-tRNA, thus extending the polypeptide by one amino acids. The second stage of the elongation cycle requires a GTPase, eukaryotic elongation factor 2 (eEF2), which enters the A-site and, through the hydrolysis of GTP, causes a change in ribosome conformation. This activates ribosome translocation to allow the next aa-tRNA to enter the A-site, thus starting a new elongation cycle.

Figure 11.33 Eukaryotic Translation Elongation Phase. This schematic represents the four basic steps of eukaryotic translation elongation. The ribosome has three tRNA-binding sites: the aminoacyl (A), peptidyl (P) and exit (E) sites. In the first step of peptide elongation, the tRNA, which is in a complex with eIF1 and GTP and contains the cognate anticodon of the mRNA coding sequence, enters the A site. Recognition of tRNA leads to hydrolysis of GTP and expulsion of eEF1 from the A site. Similarly, the deacylated tRNA at the E site is expelled. The A site and the P site tRNAs interact, allowing ribosome-catalysed peptide bond formation to occur. It involves transferring the polypeptide to aa-tRNA, thereby extending the nascent polypeptide by one amino acid. eIF5A allosterically helps form some peptide bonds, e.g. proline-proline. eEF2 then enters the A site and, through the hydrolysis of GTP, induces a change in ribosome conformation and activates translocation. The ribosome is then in the correct conformation to receive the next aa-tRNA and start another cycle of elongation.

Termination of bacterial protein synthesis occurs when a stop codon is presented at the ribosomal A-site and recognized by a class I release factor, RF1 or RF2. These release factors (RFs) have different but overlapping specificities, where RF1 reads UAA and UAG and RF2 reads UAA and UGA, with strong discrimination against sense codons. RFs are multi-domain proteins, where binding and stop codon recognition by domain 2 at the decoding site causes the universally conserved GGQ motif in domain 3 to be inserted into the A-site of the PTC. , about 80 Å from the decoding site. This event triggers hydrolysis of the peptidyl-tRNA bond at the P-site of the PTC, and the nascent peptide chain can be released through the ribosomal exit tunnel (Fig. 11.34). After peptide release, RF1 and RF2 dissociate from the post-termination complex. Dissociation is facilitated by a class II release factor called RF3, which acts as a translational GTPase that binds and hydrolyses GTP during termination.

Protein Production: A Simple Summary Of Transcription And Translation

While RF3 increases the efficiency of peptide hydrolysis, it is not an essential protein for the process. In gene knockout studies, RF3 is dispensable for the growth of Escherichia coli, and its expression is not conserved in all bacterial lineages. For example, RF3 is absent in thermophilic model organisms of the Thermus and Thermatoga genera and in the infectious Chlamydiales and Spirochaetae. This means that RF1 and RF2 can perform a complete termination phase independently of RF3 or that other GTPases from the elongation or initiation phases of translation can compensate for the action of RF3.

The release factors RF1 and RF2 acquire an open conformation (Fig. 11.34) in the 70S ribosome, which is very different from the closed conformation seen in the crystal structures of free RFs. The conformational equilibrium of free RFs in solution shows that this open conformation dominates by about 80%.

Figure 11.34. The 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 indicated by a docked ligand (red).

During peptide hydrolysis, RF factors cause rotational and conformational changes within the ribosome that allow the binding of a ribosome recycling factor (RRF) and the EF-G GTPase, leading to the separation of the large subunit from the small subunit and the release. of mRNA (Fig. 11.35).

Protein Synthesis Steps

Figure 11.35 Completion of Translation. When a 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 cause dissociation of the RF factors and conformational changes in the ribosome structure. The sequential binding of the ribosome recycling factor, RRF, and EF-G causes the dissociation of the large and small ribosomal subunits and the release of mRNA.

In eukaryotes and archaea, on the other hand an omnipotent RF reads all three stop codons. Although the translation termination mechanism is similar, there is no sequence or structural homology between bacterial RF and eukaryotic eRF1, except for the conserved all-GGQ motif required for peptide hydrolysis from tRNA. the erf3

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