Translational proofreading

this slide explains the importance of proof reading in translation and what is translational proofreading

1. TRANSLATIONAL PROOFREADING VISHNUPRIYA.C I MSc BIOCHEMISTRY

2. TRANSLATION PROCESS Cellular proteins are all synthesized by the ribosome, a conserved macromolecular machine that translates the genetic material encoded in messenger RNA (mRNA ). The ribosome consists of multiple protein and RNA components that coordinate among themselves to regulate translation.

3. In addition to the small and large ribosomal subunits bound to the mRNA, the translation machinery consists of transfer RNAs ( tRNAs ) that recognize specific codons to incorporate the respective amino acids. The whole process is also mediated by protein factors that regulate the four stages of translation: initiation, elongation, termination, and recycling.

4. STEPS INVOLVED IN TRANSLATION

5. Transfer rna and selection Translation elongation begins with decoding one mRNA codon, where a cognate aminoacylated tRNA is selected and accommodated into the ribosome for the peptidyl transfer reaction The translation apparatus has evolved to balance fidelity and efficiency during the last step of genetic information transfer, and structural dynamics and chemical energy expenditures are at the heart of the decoding machinery design .

6. Translational decoding is the process to select cognate (for an A‐site codon) tRNA substrates from the pool of different tRNA species with high accuracy (error rate of 10−3 to 10−4)5-7 and rates (5–20 amino acids per second ). DECODING PROCESS The decoding process can be divided into two phases: initial selection and proofreading. The use of structural and single‐molecule methods unveiled a structural aspect of the initial selection mechanism.

7. Two‐step kinetic proofreading model of tRNA accommodation. Initial selection of aa‐ tRNA EF‐Tu GTP ternary complex in the A site ( i ) presents the first chance of rejection of the complex ( kd ), followed by GTP hydrolysis by EF‐Tu ( ii) as the first commitment step. The second chance of rejection of aa‐ tRNA EF‐Tu GDP ternary complex (q1) occurs after GTP hydrolysis, followed by the second commitment step of EF‐Tu GDP dissociation

8. iii). One last chance of rejection of the aa‐ tRNA happens afterwards (q2), followed by accommodation of the aa‐ tRNA (iv) and the final commitment step of peptidyl transfer . The endpoint of proofreading is the peptidyl transfer reaction, in which the α‐N nucleophile of the A‐site aa‐ tRNA attacks the ester carbonyl carbon of the peptidyl‐ tRNA bound to the peptidyl‐ tRNA binding site (P site) to form a peptide bond.

9. This key chemical step in protein synthesis occurs in the peptidyl transferase center (PTC) of the large ribosomal subunit. Structural , biochemical, and computational studies revealed that there were no proteins contributing to the catalysis of peptidyl transfer in the PTC and thus the PTC was regarded as a ribozyme

10. The ultimate commitment to the incorporated aa‐ tRNA is marked by a successful peptidyl transfer reaction, which enables EF‐G to bind and begin a translocation process, where the next codon can be brought into the ribosome for the subsequent round of decoding . proofreading accuracy can be measured as the number of GTP hydrolyzed by EF‐Tu per peptide bond formation, or be calculated by dividing the overall peptide bond formation accuracy by the initial selection accuracy.

11. By comparing three different tRNA isoacceptors in reading their cognate and all near‐cognate codons demonstrated that proofreading and initial selection accuracies are positively correlated at high initial selection. Interestingly, at low initial selection, proofreading accuracy does not decrease further . Proofreading occurs in mRNA translation for protein synthesis. In this case, one mechanism is release of any incorrect aminoacyl-t RNA before peptide bond formation

12. The error rate of translation is approximately one incorrect amino acids inserted per 2000 residues QUALITY CONTROL MECHANISMS One mechanism of quality control over translational is a type of proof reading that takes place at A site . In the elongation process the charged t –RNA comes to the A site bound with the molecule of Ef -la –GTP.

13. When the anticodon of the charged t-RNA to fit tightly into the active sites to promote the formation of peptide bonds. When the codon and anticodon does not match however hydrolyses of GTP is reduced by a factor of 5x104 which allows enough time for the incorrect charged t-RNA to diffuse away and replaced with correct one. This type of proofreading at A site is called as kinetic proofreading

14. A second mechanism of quality control takes place at the P site. At this point there is a second check to determine whether the anticodon of the t-RNA is a correct match to codon in m-RNA If the match is correct, elongation proceeds normally . If there is mismatch however it means the last amino acid incorporated into the polypeptide chain is incorrect

15. When there is a mismatch at P site , a slight change in configuration of the ribosome perturbs the fidelity of the t-RNA selection at the A site. One possible outcome of the perturbation is that release factor-2(RF-2) can gain access to A site and cause premature termination even though no termination codon is present This type of translational termination due to mis -incorporation error is greatly enhanced by the presence of release factor-3 (RF-3)

16. Another possible outcome is that translation continues, and when this happens the perturbed A site makes it much more likely that another incorporation error will occur. When there are two adjacent incorporation errors the release factor are afforded even easier access to ribosome and in this probability of chain termination is great as 50%.