Role of the extra G-C pair at the end of the acceptor stem of tRNA^Hb in aminoacylation

All sequenced histidine tRNAs have one additional nucleotide at the 5′ end compared with other tRNA species. To Investigate the role of this unique structure in aainoacylation, we constructed in vitro transcripts corresponding to the E. coli histidine tRNA sequence and its variants at the G_-1-C₇₃ base pair, by using T7 RNA polymerase transcription systm. A transcript having a wild-type sequence with no modified bases was a good substrate for histidyl-tRNA synthetase (HisRS), and aminoacylation activity was affected by introduction of a triphosphate at the 5′ terminus. Base replacements at position 73 caused a marked decrease of Vmax, and deletion and substitution of the G_-1 had a remarkable effect on the aminoacylation. A mutant having an A_-1-U₇₃ pair was also not a good substrate for HisRS. Comparison among G_-1-deficient mutants showed that A was preferable rather than C as the base at position 73. These data demonstrate that the set of the G_-1-C₇₃ pair at the end of the acceptor stem of histidine tRNA is crucial for the catalytic process of aminoacylation.