Hostname: page-component-7c8c6479df-xxrs7 Total loading time: 0 Render date: 2024-03-28T15:04:14.514Z Has data issue: false hasContentIssue false

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

Published online by Cambridge University Press:  11 January 2002

SUSANNE T. NONEKOWSKI
Affiliation:
Interdepartmental Program in Medicinal Chemistry, College of Pharmacy, University of Michigan, Ann Arbor, Michigan 48109-1065, USA
GEORGE A. GARCIA
Affiliation:
Interdepartmental Program in Medicinal Chemistry, College of Pharmacy, University of Michigan, Ann Arbor, Michigan 48109-1065, USA
Get access

Abstract

In eubacteria, the biosynthesis of queuine, a modified base found in the wobble 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 transglycosylase (TGT), exchanges the genetically encoded guanine at position 34 with a queuine precursor, preQ1. 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 second 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 structural context. To further characterize the ability of TGT to recognize the U-G-U sequence in alternate contexts, we constructed mutants of the previously characterized E. coli tRNATyr minihelix. The U-G-U sequence was shifted to various positions within the anticodon loop of these mutants. Characterization of these analogs demonstrates that in addition to the normal U33G34U35 position, TGT can also recognize the U34G35U36 analog (UGU+1). The other analogs were not active. This indicates that the recognition of the U-G-U sequence is not strictly dependent upon its position relative to the stem. In E. coli, the full-length tRNA with a U34G35U36 anticodon sequence is one of the isoacceptors that codes for threonine. We found that TGT is able to recognize tRNAThr(UGU) but only in the absence of a uridine at position 33. U33, 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 ability to recognize U34G35U36, 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.

Type
Research Article
Information
RNA , Volume 7 , Issue 10 , October 2001 , pp. 1432 - 1441
Copyright
2001 RNA Society

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)