Rationale for the evolutionary retention of two unrelated lysyl-tRNA synthetases

by 1976- Ataide, Sandro Fernandes

Lysine insertion during coded protein synthesis requires lysyl-tRNALys, which is synthesized by lysyl-tRNA synthetase (LysRS). Two unrelated forms of LysRS are known: LysRS2, which is found in eukaryotes, most bacteria and a few archaea, and LysRS1, which is found in most archaea and a few bacteria. A detailed comparison of the amino acid recognition strategies of LysRS1 (Borrelia burgdorferi) and LysRS2 (Escherichia coli) was undertaken by studying the effects of lysine analogues on the aminoacylation reaction in vitro and in vivo. Also, based on comparisons of crystal structures and discrimination of lysine analogues by both LysRSs, the roles of the key residues in the active site of LysRS2 (lysS encoded) from E. coli were determined in vitro and in vivo. The differences in resistance to naturally occurring non-cognate amino acids suggest the distribution of LysRS1 and LysRS2 contributes to quality control during protein synthesis. LysRS1 and LysRS2 are not normally found together within one organism. From more than 250 publicly available genome sequences, the only instances where both LysRS1 and LysRS2 are found together are the Methanocarcineae in the archaea and certain Bacilli among the bacteria. It was shown that Methanosarcina barkeri LysRS1 and LysRS2 can together aminoacylate the rare tRNAPyl species, although the role of this ii in vitro activity remains unclear in vivo. In the pathogen Bacillus cereus, both forms of LysRS are also encoded, but genome sequence analysis does not identify tRNAPyl or any other components of the pyrrolysine insertion pathway. To investigate what role the two LysRSs might play in B. cereus, their RNA substrate specificities were investigated. It was found that in B. cereus the two different LysRSs together aminoacylate a small RNA of unknown function named tRNAOther, and that the aminoacylated product stably binds translation elongation factor Tu. In vivo analyses revealed that the class 2 LysRS was present both during and after exponential growth, while the class I enzyme and tRNAOther were predominantly produced during stationary phase. Aminoacylation of tRNAOther was also found to be confined to stationary phase, suggesting a role for this non-canonical tRNA in growth phase-specific protein synthesis. Analysis of the non-canonical Watson- Crick base pairs and a bulge in the acceptor stem of tRNAOther, present in the predicted secondary structure of tRNAOther, indicate the importance of these identity elements in recognition by the LysRS1:LysRS2 complex. The role of tRNAOther in B. cereus 14579 was investigated by the construction of a deletion strain. Comparison of the deletion strain with B. cereus wild type (wt) indicates that tRNAOther is not an essential gene but its absence de-regulates both the production of a Bactericin-Like Inhibitory Substance (BLIS) and the macrolide efflux protein conferring resistance against other bacterial macrolides. Also, other secondary metabolic effects were observed such as loss of resistance against certain compounds in the deletion strain. These results implicate tRNAOther in multiple regulatory functions that remain, as yet, uncharacterized. iii