Exploring the limits of tRNA substrate recognition by <italic>Escherichia coli</italic> tRNA -guanine transglycosylase.

Abstract

The posttranscriptional tRNA modification enzyme, tRNA-guanine transglycosylase (TGT) is one of the key enzymes involved in the biosynthesis of the hypermodified base, queuine, in eubacteria. Previous studies have identified a minimal positive RNA recognition motif for <italic>Escherichia coli</italic> TGT consisting of a stable minihelix that contains a U₃₃-G₃₄-U₃₅ sequence in a 7-base loop that mimics the anticodon loop sequence of queuine-cognate tRNAs. Recently, it was discovered that TGT was capable of recognizing <italic>in vitro</italic> transcribed yeast tRNA<super>Phe </super>, despite the fact that it lacks this sequence. However, the precise site of activity in this tRNA was not clear. Mutagenesis studies indicate that G₅₃ is the guanine responsible for activity with TGT since substitution of G₅₃ eliminates TGT activity and converts a substrate into an inhibitor. To further characterize the ability of TGT to recognize the U-G-U sequence in alternate contexts, mutants of the previously characterized <italic>E. coli</italic> tRNA<super>Tyr</super> minihelix were evaluated. Characterization of these analogs demonstrates that TGT can also recognize a U₃₄G₃₅U₃₆ sequence. Furthermore, this recognition translates to full length tRNA but only in the absence of a uridine at position 33. U₃₃ has previously been shown to strongly influence the conformation of the anticodon loop of certain tRNAs. Mutation of this base confers upon TGT the ability to recognize U₃₄G₃₅U₃₆ in <italic> E. coli</italic> tRNA<super>Thr(UGU)</super> and suggests that loop conformation affects recognition. The fact that the other analogs were not active indicates that, although TGT is capable of recognizing the U-G-U sequence in additional contexts, this recognition is not indiscriminate. To probe the role of the 2<super>'</super>-hydroxyl in recognition, 2<super>'</super>-deoxyribose analogs that contain deoxyuracil in place of thymidine were examined. There was very little difference between the kinetic parameters of the minihelix RNAs and their deoxyribose counterparts. This demonstrates that none of the 2<super>'</super>-hydroxyls are required for recognition or catalysis by TGT. The results from this work illustrate that <italic>E. coli</italic> TGT can recognize other RNAs and DNAs <italic>in vitro</italic> and possibly even <italic> in vivo</italic>. Thus, the physiological implications of queuine incorporation may encompass more than its incorporation into tRNA.