tRNA recognition by tRNA-guanine transglycosylase from Escherichia coli: The role of U33 in U-G-U sequence recognition

In eubacteria, the biosynthesis of queuine, a modified base found in the wo bble position (#34) of tRNAs coding for Tyr, His, Asp, and Asn, occurs via a multistep pathway. One of the key enzymes in this pathway, tRNA-guanine t rans-glycosylase (TGT), exchanges the genetically encoded guanine at positi on 34 with a queuine precursor, preQ(1). Previous studies have identified a minimal positive RNA recognition motif for Escherichia coli TGT consisting of a stable minihelix that contains a U-G-U sequence starting at the secon d position of its seven base anticodon loop. Recently, we reported that TGT was capable of recognizing the U-G-U sequence outside of this limited stru ctural context. To further characterize the ability of TGT to recognize the U-G-U sequence in alternate contexts, we constructed mutants of the previo usly characterized E. coli tRNA(Tyr) minihelix. The U-G-U sequence was shif ted to various positions within the anticodon loop of these mutants. Charac terization of these analogs demonstrates that in addition to the normal U(3 3)G(34)U(35) position, TGT can also recognize the U(34)G(35)U(36) analog (U GU(+1)). The other analogs were not active. This indicates that the recogni tion of the U-G-U sequence is not strictly dependent upon its position rela tive to the stem. In E. coli, the full-length tRNA with a U(34)G(35)U(36) a nticoclon sequence is one of the isoacceptors that codes for threonine. We found that TGT is able to recognize tRNA(Thr(UGU)) but only in the absence of a uridine at position 33. U-33, an invariant base present in all tRNAs, has been shown to strongly influence the conformation of the anticodon loop of certain tRNAs. We find that mutation of this base confers on TGT the ab ility to recognize U(34)G(35)U(36), and suggests that loop conformation aff ects recognition. The fact that the other analogs were not active indicates that although TGT is capable of recognizing the U-G-U sequence in addition al contexts, this recognition is not indiscriminate.