Change of tRNA identity leads to a divergent orthogonal histidyl-tRNA synthetase/tRNAHis pair

Abstract

Mature tRNAHis has at its 5′-terminus an extra guanylate, designated as G−1. This is the major recognition element for histidyl-tRNA synthetase (HisRS) to permit acylation of tRNAHis with histidine. However, it was reported that tRNAHis of a subgroup of α-proteobacteria, including Caulobacter crescentus, lacks the critical G−1 residue. Here we show that recombinant C. crescentus HisRS allowed complete histidylation of a C. crescentus tRNAHis transcript (lacking G−1). The addition of G−1 did not improve aminoacylation by C. crescentus HisRS. However, mutations in the tRNAHis anticodon caused a drastic loss of in vitro histidylation, and mutations of bases A73 and U72 also reduced charging. Thus, the major recognition elements in C. crescentus tRNAHis are the anticodon, the discriminator base and U72, which are recognized by the divergent (based on sequence similarity) C. crescentus HisRS. Transplantation of these recognition elements into an Escherichia coli tRNAHis template, together with addition of base U20a, created a competent substrate for C. crescentus HisRS. These results illustrate how a conserved tRNA recognition pattern changed during evolution. The data also uncovered a divergent orthogonal HisRS/tRNAHis pair.