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.

10. • 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

11. 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.

12. 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

13. 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.

14.  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

17. 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.

18. • 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

19. • 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

20. • 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)

21. • 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%.