3. In vivo assay system for genetic analysis of initiation
To develop a plasmid based assay system for in vivo analyses of the structure function relationship of i-tRNA, chloramphenicol (Cm) acetyltransferase (CAT) gene was mutated to encode CATam1 mRNA possessing UAG as initiation codon (in place of AUG) along with two other mutations at codons 2 and 5 to avoid background initiation (Fig. 2B, i) (Varshney and RajBhandary 1990). A corresponding change was also made in i-tRNA gene (metY ) to encode an i-tRNA with CUA anticodon (i-tRNACUA) to allow it to pair with the UAG initiation codon in CATam1 mRNA.E. coli harbouring CATam1 gene (on a plasmid, pCATam1) gained resistance to Cm (CmR) only if it also simultaneously possessed i-tRNA gene (metY CUA) to encode i-tRNACUA. The pCATam1metY CUA (harbouring both the CATam1 and metY CUAgenes) provided an assay system for in vivo initiation (Fig. 2B, i and ii ). Any mutants of i-tRNACUAthat continue to confer CmR to E. coli , are functional as initiators; and those that are Cm sensitive (CmS) are non-functional as initiators. In this assay system, initiation occurred predominantly with fGln rather than with fMet (the CAU to CUA change in the anticodon, the mutant i-tRNACUA becomes highly deficient for recognition by MetRS but it becomes a good substrate for GlnRS (Schulman and Pelka 1985; Seong et al. 1989)). Gln attached to i-tRNA is also an excellent substrate for Fmt, an enzyme that formylates the α-amino group of most amino acids attached to i-tRNA (Mayer et al. 2003). Importantly, the assay system showed that even though Met is evolutionarily conserved as an initiating amino acid, initiation can occur with other amino acids even in vivo . Further, the assay system showed that when the mismatch at 1-72 position is converted to a Watson-Crick pair, the mutant i-tRNA becomes a dual function tRNA that participates both at the initiation and elongation steps (Govindan, Ayyub, and Varshney 2018; Varshney et al. 1991). When the special features of i-tRNA found in its acceptor stem and anticodon stem were transplanted into elongator tRNAs, they gained initiator function (Varshney et al. 1993). Not surprisingly, when the i-tRNA 3GC pairs in the anticodon stem were changed with those found in the elongator tRNAMet(U29-A41, C30-G40, A31-ᴪ39 or 3GC mutant i-tRNA or ua/cg/au; mutations shown in small letters), the i-tRNA mutant failed to function in initiation (Fig. 2B, iii) despite its efficient aminoacylation and formylation (Das et al. 2008; Mandal et al. 1996; Shah et al. 2019).