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Translation termination in bacteria involves precise reading of stop codons (UAA, UAG, UGA) and coordinated peptidyl-tRNA hydrolysis by the class-I release factors (RFs) on the ribosome (70S). This thesis investigates the evolutionary and post-translational modification mechanisms of these RFs and the concurrent effect on bacterial translation termination.

Unlike eukaryotes with a single RF, bacteria have two RFs. Release factor 1 (RF1) reads UAA and UAG; release factor 2 (RF2) reads UAA and UGA codons. So, why do bacteria host two RFs? To answer this, we performed an in vivo evolution experiment to explore how RF2 evolution compensates for the loss of RF1. Characterization of the evolved RF2 mutants, specifically E167K RF2, using both in vivo and in vitro peptide release assay reveals its ability to read the RF1-specific UAG codon and also the tryptophan (UGG) codon, displaying a functional trade-off termed “collateral toxicity”. Further, fast-kinetics-based peptide release assay shows that E167K RF2 is generally efficient in peptide release on UAA and UGA but significantly more efficient on UAG and UGG than WT RF2. This increased efficiency is primarily due to the higher affinity of E167K RF2 to the 70S. Our 2.8 Å cryo-EM structures demonstrated K167 to be engaged in hydrogen bond interactions with the rRNA, that are absent in WT RF2 having E167. Further, the mutant displays somewhat destabilized conformation when unbound, bypassing the conformational change check-point and facilitating the reading of near-cognate UAG and UGG codons. 

Post-translational methylation on the conserved GGQ motif of RF1/2 increases the efficiency of translation termination, but its role in termination accuracy was unknown. We compared the methylated and unmethylated variants of RF1/2 for cognate and near-cognate codon recognition. The unmethylated RFs exhibited lower termination accuracy, likely caused by the loss of conformational stability in the absence of GGQ methylation.

In summary, these studies reveal the compensatory evolution of E167K RF2 as a tighter binder of the ribosome with the destabilized compact conformation that enhances UAG reading at the expense of UGG reading. Additionally, our study shows that GGQ methylation maintains the conformational stability of RF2 and facilitates accurate stop codon recognition.