tRNA-guanine transglycosylase from Escherichia coli: Recognition of noncognate-cognate chimeric tRNA and discovery of a novel recognition site withinthe T Psi C arm of tRNA(Phe)

tRNA-guanine transglycosylase (TGT) is a key enzyme involved in the posttra nscriptional modification of tRNA across the three kingdoms of life. In euk aryotes and eubacteria, TGT is involved in the introduction of queuine into the anticodon of the cognate tRNAs. In archaebacteria, TGT is responsible for the introduction of archaeosine into the D-loop of the appropriate tRNA s. The tRNA recognition patterns for the eubacterial (Escherichia coli) TGT have been studied. These studies are all consistent with a restricted reco gnition motif involving a U-G-U sequence in a seven-base loop at the end of a helix. While attempting to investigate the potential of negative recogni tion elements in noncognate tRNAs via the use of chimeric tRNAs, we have di scovered a second recognition site for the E. coli TGT in the T Psi C arm o f in vitro-transcribed yeast tRNA(Phe). Kinetic analyses of synthetic mutan t oligoribonucleotides corresponding to the T Psi C arm of the yeast tRNA(P he) indicate that the specific site of TGT action is G53 (within a U-G-U se quence at the transition of the T Psi C stem into the loop). Posttranscript ional base modifications in tRNAPhe block recognition by TGT, most likely d ue to a stabilization of the tRNA structure such that G53 is inaccessible t o TGT These results demonstrate that TGT can recognize the U-G-U sequence w ithin a structural context that is different than the canonical U-G-U in th e anticodon loop of tRNA(Asp). Although it is unclear if this second recogn ition site is physiologically relevant, this does suggest that other RNA sp ecies could serve as substrates for TGT in vivo.