Question

6. (2 pts) Why do you think a mutation that lowers the effectiveness of IF3 might lower the overama protein synthesis in an E. coli cell? wnat step in translation would most likely be affected -initiation, elongation, or terminanon (2 pts) In an experiment, you purify a cysteine-charged tRNA and chemically alter the amino acid attached to it converting it to alanine. You place this charged tRNA in a cell-free protein-synthesizing system with all of the other charged tRNAs, mRNA and other substances needed for the synthesis of proteins. What if anything, will be different from the normal protein coded for by the mRNA in the mixture? How does this affect its function?

Answer 1

IF3 is one of the three factors required for the initiation of protein synthesis. In Escherichia coli, IF3 is a protein of 180 amino acids encoded by infC. It is an RNA-binding protein.

IF3 is involved in the fidelity of translation initiation at several levels, including start-codon discrimination, mRNA translation, and initiator-tRNA selection. The IF3 C-terminal domain (CTD) is required for binding to the 30S ribosomal subunit. N-terminal domain (NTD) function is less certain but likely contributes to initiation fidelity. Point mutations in either domain can decrease initiation fidelity, but C-terminal domain mutations may be indirect

Functions of IF3 can be explained in detail as follows :

1. antagonizes the association between 30S and 50S subunits, thus supplying the pool of free 30S subunits required for translation initiation

2. accelerates the on-rate of codon–anticodon interaction at the P-site, thus stimulating the formation of 30S initiation complexes

3. promotes the rapid dissociation of fMet-tRNA from initiation complexes (formed at the 5′ AUG triplet of leaderless mRNAs) as well as the dissociation of pseudo-initiation complexes containing aminoacyl-tRNAs other than initiator fMet-tRNA or initiation complexes containing fMet-tRNA and triplets other than the initiation triplets AUG, GUG, and UUG

4. as a probable consequence of (ii) and (iii), IF3 ensures the efficiency and fidelity of initiation site selection inhibition, if present in stoichiometric excess over the ribosomes

5. IF3 induces an adjustment of the mRNA, which is shifted from the ‘stand-by site’ to the ‘P-decoding site’ on the 30S ribosomal subunit.

So, mutation lowering IF3 effectiveness will affect the above-said functions, lowering the overall rate of protein synthesis in E. coli and will affect the initiation step of translation.

An aminoacyl-tRNA synthetase is an enzyme that attaches the appropriate amino acid onto its tRNA forming aminoacyl tRNA. This is called charging or loading of tRNA. During protein synthesis, the ribosome transfers this amino acid from charged tRNA onto the growing peptide, according to the genetic code. Aminoacyl tRNA, therefore, plays a major role in RNA translation leading to the formation of specific proteins.  

Proteins perform many essential physiological functions, including catalyzing biochemical reactions, synthesizing and repairing DNA, transporting materials across the cell, receiving and sending chemical signals, responding to stimuli and providing structural support.

Because the form determines function, any slight change in protein's shape can make protein dysfunctional. Small changes in the amino acid sequence of a protein can cause devastating genetic diseases.

So, to conclude, if tRNA is charged with alanine instead of cysteine, the amino acid sequence of the resulting protein will change leading to the formation of different protein which might alter any physiological function.