(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.