(i) Role of i-tRNA in translation initiation and ribosome
biogenesis.
(a) Initiator tRNA gene copy numbers and its abundance in
the fidelity of initiation: E. coli possesses four copies of i-tRNA
genes. Of these, three are located at 63.5’ as metZWV operon
(tRNAfMet1); and the fourth one, metY(tRNAfMet2) is located at 71.5’ (Kenri, Imamoto, and
Kano 1994). All four copies of i-tRNAs in E. coli B-strains are
identical (Mandal and RajBhandary 1992). However, in the K-strains,
tRNAfMet2 harbours an A at position 46, whereas other
i-tRNAs (tRNAfMet1) harbour m7G at
this position (m7G46). The
significance of retention of i-tRNAs with
m7G46 and A46 inE. coli K strains is unclear. Nonetheless, either of the loci is
sufficient for survival of E. coli (Kenri, Imamoto, and Kano
1992; Tsuyoshi et al. 1991). The cellular abundance of i-tRNA is
proportional to the copy number of i-tRNA genes (Kapoor, Das, and
Varshney 2011). Deletion of metZWV reduces E. coli growth
and confers cold sensitivity phenotype to it. However, deletion ofmetY has almost no effect on the growth of the strain. Samhita
and colleagues found that E. coli strains with four i-tRNA genes
outcompeted a strain with three i-tRNA genes when grown in rich medium.
However, the opposite was true when they were grown in nutrient-poor
conditions for a prolonged time (Samhita, Nanjundiah, and Varshney
2014). These observations suggested that i-tRNA genes, in bacteria, may
be subjected to dynamic copy number changes. The presence of E.
coli strains with five i-tRNA genes (HS) was reported in the gut
(nutrient-rich) and with three i-tRNA genes (IAI39) was identified in
urinary tract infections (nutrient-poor). In our genetic analyses, a
severe depletion of i-tRNA in E. coli either by promoter down
mutations (as present in a series of suppressor strains identified in
our genetic screen) or by engineering a deletion of the entiremetZWV locus (Kapoor et al. 2011), revealed a link between i-tRNA
abundance and the fidelity of initiation. Depletion of cellular i-tRNA
allowed initiation not only with the 3GC mutant i-tRNA and the
noncanonical i-tRNAs lacking a full complement of 3GC pairs in the
anticodon stem, but also with elongator tRNAs (Fig. 3, ii )
(Kapoor et al. 2011; Samhita et al. 2013). Thus, a high abundance of
canonical i-tRNAs is required not only to overcome the rate-limiting
step of translation initiation (Gualerzi and Pon 1990; Laursen et al.
2005) but also to discriminate against binding of ”i-tRNA like” or
elongator tRNAs in the P-site (Kapoor et al. 2011; Samhita et al. 2013).
These observations allowed us to then engineer the growth of E.
coli exclusively on i-tRNAs lacking either the first GC pair, the third
GC pair or both the first and the third GC pairs as found in some
mycoplasma and rhizobium species (Samhita et al. 2012). In a recent
study where all the components of the translation machinery and entire
translatome of the human pathogen Mycoplasma pneumoniae(Mpn ) were analysed, the abundance of i-tRNAs was found to be
massive at 12.1% as opposed to ~3% in E. coli(Dong, Nilsson, and Kurland 1996; Weber et al. 2023). Given that the
anticodon stem of i-tRNA in Mpn harbours AU/GC/GU sequence
instead of the canonical GC/GC/GC, the high abundance of i-tRNA could be
the organism’s way of outcompeting “i-tRNA like” or elongator tRNA
binding in the P-site, mitigating loss of initiation fidelity.
These observations raise a question if there are any natural means of
regulating fidelity of translation initiation by regulating i-tRNA
abundance in bacteria? Several lines of evidence suggest that E.
coli could regulate the levels of its i-tRNA contents depending on the
nutritional status. At least in vitro , ppGpp has been shown to
downregulate expression of metZWV during the stringent response
(Takahiro, Shunsuke, and Fumio 1988). Likewise, expression frommetY may be regulated by cAMP-CAP a global regulator of
transcription, as well as ArgR, a specific transcriptional regulator of
arginine metabolism (Krin et al. 2003). Moreover, studies have shown
that if E. coli is deprived of leucine, the level of
aminoacylated i-tRNAs decreases dramatically (Dittmar et al. 2005).
Another mechanism of downregulating i-tRNA levels is based on the action
of VapC toxin of the VapBC toxin-antitoxin module in Shigella
flexneri 2a and the VapCLT2 of Salmonella enterica serovar Typhimurium
LT2, both of which are site-specific tRNases that target 3GC pairs in
the i-tRNA anticodon stem (Winther and Gerdes 2011). Based on our
findings, such a depletion of i-tRNA will favour initiation with
unconventional i-tRNAs or elongator tRNAs. In yeast, i-tRNA depletion
prompts translation of GCN4, a nutritional stress transcription factor
(Conesa et al. 2005). Further, the levels of initiator and elongator
methionine tRNAs are negatively associated with cell proliferation
versus quiescence (Kanduc 1997). Thus, it seems that downregulating
i-tRNA levels could be a cellular response to overcome stress by
promoting ‘leakiness’ in the translation apparatus.
(b) Initiator tRNA abundance and ribosome maturation:
Another consequence of i-tRNA depletion we observed in E. coliwas the gain of cold sensitive phenotype in the strains deleted formetZWV . We showed that the i-tRNA, more specifically its 3GC
pairs, play a crucial role in the terminal stages of ribosome maturation
by prompting trimming of the extra sequences at the 3’ and 5’ ends of
the 17S precursor to produce mature 16S rRNA during the pioneering round
of initiation (Fig. 4) . Based on the genetic interactions, the
extra sequences may be trimmed by RNase R, RNase II, and RNase PH
(Samhita et al. 2012; Shetty and Varshney 2016; Tsuyoshi et al. 1991).
More recently, based on the analysis of lamotrigine toxicity (which
targets IF2), we showed that this role of i-tRNA in maturation of 16S
rRNA is mediated through IF2 and i-tRNA complex bound to the 30S. Also,
lamotrigine mediated inhibition of ribosome biogenesis led to an
increased accumulation of ribosome binding factor A (RbfA), a late stage
ribosome biogenesis factor, on 30S (Singh et al. 2023).
In other investigations, we noted that overexpression of i-tRNA rescues
biogenesis defects resulting from the deficiency of methylations at G966
and C967 in 16S rRNA in the P-site or deletion of the C-terminal
residues of uS9 (S126, K127 and R128, or SKR) that impact i-tRNA binding
(Arora, Bhamidimarri, Bhattacharyya, et al. 2013; Ayyub et al. 2018).
The binding of i-tRNA may affect the 3′ end region of 16S rRNA through
conserved residues, for example, G1338 and A1339, which interact with
the 3GC pairs for the accuracy of i-tRNA selection in initiation. The
conformational changes induced in the ribosome during initiation might
then signal the final processing of the 17S rRNA to 16S rRNA.