Research Projects

Translation initiation from cellular mRNAs uses a special tRNA, the initiator tRNA (i-tRNA) which is naturally aminoacylated with methionine (Met) using methionine tRNA synthetase (MetRS). Thus, translation is predominantly initiated with Met (fMet in bacteria and eukaryotic organelles) in all domains of life, irrespective of the identity of the initiation codons. Paradoxically, a majority of the proteins lose the initiator Met/fMet during their maturation, questioning the evolutionary importance of Met as the initiating amino acid. Earlier, we showed initiation with non-methionine amino acids in orthogonal systems of plasmid-based reporters. In this project, to address the evolutionary question of why the exclusive use of Met in initiation, to generate a system of unconventional means of initiation with non-methionine amino acid, to generate a new approach to investigate antimicrobial resistance (AMR), and to expand our toolkit of recombinant protein production for research and biotechnology, we propose to initiate translation with Ala, both in a plasmid-based reporter system; and on a global scale in Escherichia coli. The choice of Ala for initiation is based on various considerations including, (i) prevalence of Ala at the N-terminal of mature proteins, and (ii) our preliminary observation of the technical feasibility of engineering the identity switch in i-tRNAs for recognition by AlaRS. A C3:G70 to G3:U70 mutation in the acceptor stem of i-tRNA has sufficed for its aminoacylation with Ala. For global initiation with Ala, once an identity switch in i-tRNA (encoded from a plasmid-borne gene) for aminoacylation with Ala and its formylation is made robust, the next step is to delete all four of the naturally occurring tRNAfMet genes (metZ, metW, metV and metY) from E. coli chromosome using a technology already established in our laboratory.