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).