Use of semi-synthetic transfer RNAs to probe molecular recognition by Escherichia coli proline-tRNA synthetase

Background: The attachment of specific amino acids to the 3′-end of cognate transfer RNAs (tRNAs) is catalyzed by a class of enzymes known as aminoacyl-tRNA synthetases (aaRS).We have previously demonstrated that Escherichia coli proline-tRNA synthetase (ProRS) can aminoacylate semi-synthetic RRNAs prepared by annealing two RNA oligonucleotides. We set out to examine the factors that are important in selective recognition of tRNA Pro by ProRS, using semi-synthetic RRNAs and full-length tRNA transcripts. Results: Deletion of nucleotides A58, A59, and U60 in the TΨC-loop of semi-synthetic RRNAs has no adverse effect on aminoacylation. Nucleotide deletions that extend into the TΨC stem, particularly beyond C61, significantly reduce the efficiency of aminoacylation, however. Site-directedmutagenesisof full-length tRNA Pro transcripts shows that, although there is no strict sequence requirement at base pair 52·62 in the TΨC stem, helix destabilizing purine-purine mismatches at this position result in decreased aminoacylation activity. Moreover, aminoacylation is severely affected when a DNA-RNA hybrid helix is incorporated into the acceptor-TTC stem domain. Conclusions: At least three nucleotides in the TΨC-loop are dispensable for aminoacylation of E. coli tRNA Pro. These results, combined with previous data, demonstrate that four out of five of the so-called ‘variable pocket’ nucleotides are not important for recognition of tRNA Pro by E. coli ProRS. ProRS is also sensitive to changes that are likely to alter the helical conformation in the TΨC stem.