The primary interest of the lab is to study mechanistic aspects of protein synthesis and DNA repair using E. coli and Mycobacteria as model systems. The lab has two main research groups: The Protein Synthesis Group and The DNA Repair Group.

Protein Synthesis

  • Mechanism of ribosome recycling:Subsequent to the action of release factors at the step of termination, ribosomes remain bound to the mRNA in a post-termination complex. In eubacteria, the post-termination complexes are disassembled by the action of ribosome recycling factor (RRF) and elongation factor G (EFG). We have shown that specific interactions between RRF and EFG are essential to recycle the post-termination complexes. Such interactions between RRF and EFG are also required to recycle the stalled ribosomes (pre-termination ribosomal complexes) during the step of elongation by releasing peptidyl-tRNAs from them. The function of EFG in ribosome recycling is different from its classical role in translocation, and our recent observations suggest that a distinct set of interactions of EFG with RRF and the ribosome is crucial in this process. Further, we have shown that there is a functional interaction between RRF and IF3 in recycling of both the pre-, and post-termination ribosomal complexes. Present research is focused on the interplay of various factors during the various steps of protein synthesis.
  • The structure-function relationship of the  coliinitiator tRNA : Eubacterial initiator tRNAs (tRNAfMet) possess unique features such as a mismatch at the top of the acceptor stem and the three consecutive G, C base pairs in the anticodon stem. The latter feature which is highly conserved in all the three kingdoms of life has been implicated in preferential binding of tRNAfMet to the ribosomal P-site. How this feature is exploited by ribosomes in selecting tRNAfMethas been a long standing question. We have isolated several E. coli strains which allow initiation with tRNAs lacking the three consecutive G-C base pairs. In one of the strains, a severe deficiency of methionine and S-adenosyl-L-methionine; and lack of nucleoside methylations in rRNA allow initiation with tRNAfMetcontaining mutations in one, two or all the three G-C base pairs, and also with an elongator tRNA. Mutations in specific methyltransferases support a role for rRNA methylations in tRNAfMet selection on the ribosome. In yet another strain, reduction in levels of wildtype initiator tRNA allows initiation with a mutant tRNA lacking 3 G-C pairs, showing that there is a competition between initiator and elongator tRNAs for P-site binding. We have also shown that it is possible to sustain E. coli on a 3 G-C mutant tRNA and that the 3 G-C rule can in fact, be reduced to a middle G-C rule. Future studies are aimed at characterization of the remaining supressor strains to allow detailed understanding of the mechanism of initiation.
  • A single mammalian mitochondrial translation initiation factor functionally replaces two bacterial factors:In eubacteria, three initiation factors IF1, IF2, and IF3 are vital. We have shown that bovine mitochondrial IF2 (IF2mt) complements  coli containing a deletion of IF2 gene (infB). We find that IF1 is no longer essential in an IF2mt-supported E. coli ΔinfB strain. We suggest that a conserved insertion of 37 amino acids in the IF2mt substitutes for the function of IF1. Deletion of this insertion from IF2mt supports E. coli for the essential function of IF2. However, in this background, IF1 remains essential. The future studies are aimed at understanding of how the 37 amino acid insert in IF2mt substitutes for the function of IF1.

DNA Repair

DNA repair in mycobacteria: Owing to their G+C rich genomes, mycobacteria are naturally at increased risk of cytosine deamination (to uracil), and oxidative damage to guanosine (to 8-oxoG). The uracil and 8-oxoG damages in DNA are repaired by Ung and Fpg, respectively. In addition, nucleotide excision repair (NER) is known to repair a broad spectrum of DNA damages. We have shown that the ung- strains of M. smegmatis exhibit increased mutator phenotype and poor endurance in mouse macrophages. Using M. smegmatis strains wherein fpgmutY (encoding proteins involved in 8-oxoG repair) or uvrB (involved in NER pathway) genes have been inactivated, we have shown that the NER pathway is vital in protecting the organism from a variety of commonly encountered DNA damaging agents. Considering that in the host, the pathogen is subjected to various DNA damaging agents (RNI, ROS), a compromise in the DNA repair capacity could prove detrimental to the pathogen’s existence. We are continuing our studies in DNA repair in M. tuberculosis with the aim to develop newer drug targets and also the attenuated strain as possible vaccine candidates.

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Dr. Sudhir Singh

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Dr. Rajasree K P

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