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 of 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 tRNAs prepared by annealing two RNA oligonucleotides. We set out to examine the factors that are important in selective recognition of tRNAPro by ProRS, using semi-synthetic tRNAs and full-length tRNA transcripts.

RESULTS

Deletion of nucleotides A58, A59, and U60 in the T psi C-loop of semi-synthetic tRNAs has no adverse effect on aminoacylation. Nucleotide deletions that extend into the T psi stem, particularly beyond C61, significantly reduce the efficiency of aminoacylation, however. Site-directed mutagenesis of full-length tRNAPro transcripts shows that, although there is no strict sequence requirement at base pair 52.62 in the T psi C 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-T psi C stem domain.

CONCLUSIONS

At least three nucleotides in the T psi C-loop are dispensable for aminoacylation of E. coli tRNAPro. 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 tRNAPro by E. coli ProRS. ProRS is also sensitive to changes that are likely to alter the helical conformation in the T psi C stem.