Physiological significance of tRNA sequences and modifications in regulation of the bacterial growth

Abstract

Transfer RNA or tRNA is one of the vital components of translation machinery where it acts as an adaptor molecule to bring amino acid to the ribosome in response to the genetic code in mRNA. Its key role is to decode the codons on mRNA and bring in amino acids to the ribosome to form polypeptides. Apart from its canonical role in translation, tRNA takes part in various other cellular process. tRNA forms a secondary cloverleaf structure and L-shape tertiary structure. There are a number of post-transcriptional modifications found in tRNA which help it in maintaining translational fidelity and proper tertiary structure folding. There are two classes of tRNAs that are found in cells, one dedicated for translation initiation, known as initiator tRNA (i-tRNA) and the others involved at the step of elongation, called as elongator tRNAs. In this study I have worked on the aspect of the single nucleotide polymorphism and modifications in i-tRNA, and on the role of evolutionary conserved sequence complementarity found in elongator tRNAs using Escherichia coli as model.

Part-I: Physiological of significance two isoform of i-tRNA in bacteria

Escherichia coli possesses four initiator tRNA (i-tRNA) genes, three of which are present together as metZWV and the fourth one as metY. In E. coli B, all four genes (metZWV and metY) encode i-tRNAfMet1, in which the G residue at position 46 is modified to m7G46 by TrmB (m7G methyltransferase). However, in E. coli K, because of a single nucleotide polymorphism, metY encodes a variant, i-tRNAfMet2, having an A in place of m7G46. We generated E. coli strains to explore the importance of this polymorphism in i-tRNAs. The strains were sustained either on metYA46 (metY of E. coli K origin encoding i-tRNAfMet2) or its derivative metYG46 (encoding i-tRNAfMet1) in single (chromosomal) or plasmid borne copies. We show that the strains sustained on i-tRNAfMet1 have growth fitness advantage over those sustained on i-tRNAfMet2. The growth fitness advantages are more pronounced for the strains sustained on i-tRNAfMet1 in nutrient rich media than in nutrient poor media. The growth fitness of the strains correlates well with the relative stabilities of the i-tRNAs in vivo. Further, the atomistic molecular dynamic simulations support higher stability of i-tRNAfMet1 than that of i-tRNAfMet2. The stability of i-tRNAfMet1 remains unaffected upon deletion of TrmB. These studies highlight how metYG46 and metYA46 alleles might influence growth fitness of E. coli under certain nutrient limiting conditions.

Part-II: Evidence of regulatory role of the evolutionary conserved sequence complementarity between tRNAs

The tRNAs are non-coding RNAs (ncRNAs) known for their classical role is in decoding mRNAs. We compared all possible pairs of tRNA sequences belonging to bacterial species and sub-species. The analysis revealed pairwise similarities for different combinations of tRNA sequences. Also, we detected varied sequence complementarities between some tRNA pairs. The organisms possess varying levels of tRNAs encoded by their respective genes. We hypothesized that annealing of the two tRNAs might regulate their functions. Here, we studied the physiological significance of one such pair of E. coli tRNA genes (lysT, encoding an abundant tRNALys, and argU encoding a rare tRNAArg) showing ~80% complementarity. In vitro experiments showed that tRNALysT and tRNAArgU anneal to form a heterodimer, and the presence of DNA oligomers complementary to the interacting sequences, inhibited the heterodimerization in a concentration dependent manner. Further, while overexpression of tRNAArgU did not impact the culture growth, that of tRNALysT showed a growth inhibitory (toxic) phenotype. The tRNALysT based toxicity was enhanced when the cultures were grown at lower temperature. The toxicity of LysT overexpression is consistent with the sequestration of tRNAArgU by tRNALysT. Use of AGA minigene and hybrid phage, λimm-P22 growth on ssrA (tmRNA) mutant strains was also consistent with the sequestration of tRNAArgU and tRNALysT. In addition, we observed the presence of tRNA-derived small fragments (tRFs) formed from tRNALysT and tRNAArgU, which may also interfere with the tRNA function. Taken together, these observations support the novel hypothesis of the regulatory role of the evolutionary conserved complementarities between the two tRNAs in cellular growth.

Part-III: Does TrmA deficiency rescue the slow growth phenotype of the Fmt deficient E. coli?

tRNA modifications are important for proper functioning of the tRNA. In bacteria, initiator Met-tRNAfMet undergoes a characteristic modification known as formylation catalysed by Methionyl-tRNAfMet N-formyltransferase (Fmt) encoded by fmt gene. Fmt is important for efficient translation initiation. Another, modification found in the TΨC arm of the tRNA is 5-methyluridine (m5U54) at the 54th position is catalysed by trmA encoded m5U54-methyltransferase. Earlier report has suggested that a strain deficient in 5-methyluridine modification may facilitate formylation independent translation initiation. It is shown that m5U54 modification found in the elbow region of tRNA plays a significant role in stabilising the tertiary structure. Any perturbation in the elbow region, could impact discrimination of i-tRNAfMet selection at the ribosomal P-site by IF3 which will then compensate for the deficiency of formylation. So, we hypothesize that strain deficient with m5U54 modification may facilitate formylation independent initiation. We observed that in absence of m5U54 modification there is some change in structural conformation of i-tRNAfMet1. Interestingly, upon growth analyses, we do observe that ∆trmA-∆fmt::kan strain shows a slightly less doubling time than the fmt deleted strain. However, the growth patterns of the strains are highly heterogenous, and complementation of ∆trmA-∆fmt::kan strain with the trmA does not show any growth difference suggesting that the improved growth of the double knockouts may be due to some uncharacterised suppressor mutations.