Substrate Specificity for 4-Thiouridine Modification in Escherichia coli

Charles T Lauhon,Whitney M Erwin,Giangthy N Ton

doi:10.1074/jbc.m401757200

Charles T Lauhon, Whitney M Erwin + Show 1 more

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https://doi.org/10.1074/jbc.m401757200

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Abstract

The biosynthesis of 4-thiouridine (s4U) in Escherichia coli tRNA requires the action of both the thiamin pathway enzyme ThiI and the cysteine desulfurase IscS. IscS catalyzes sulfur transfer from l-cysteine to ThiI, which utilizes Mg-ATP to activate uridine 8 in tRNA and transfers sulfur to give s4U. In this work, we show through deletion analysis of unmodified E. coli tRNA(Phe) that the minimum substrate for s4U modification is a mini-helix comprising the stacked acceptor and T stems containing an internal bulged region. The size of the bulged loop must be at least 4 nucleotides and contain the target uridine as the first nucleotide. Replacement of the T loop sequence with a tetraloop in the deletion substrate increases activity and shows that the TpsiC primary sequence is not a recognition element. An unmodified tRNA(Phe) transcript in which the 3'-terminal ACCA sequence is removed to give a blunt terminus has <0.1% activity, although the addition of a single overhanging base essentially restores activity. In addition, reducing the distance of the 3' terminus relative to U8 by as little as 1 bp severely impairs activity. By dissecting a minimal RNA substrate in the T loop region, a two-piece system consisting of a substrate RNA and a "guide" RNA is efficiently modified. Our results indicate that outside of the modified U8, there is no primary sequence requirement for substrate recognition. However, the secondary and tertiary structure restrictions appear sufficient to explain why s4U modification is limited in the cell to tRNA.

Highlights

All organisms chemically modify the bases of their tRNA [1, 2]
We show through deletion analysis of unmodified E. coli tRNAPhe that the minimum substrate for s4U modification is a mini-helix comprising the stacked acceptor and T stems containing an internal bulged region
The assay was measured for the incorporation of 35S from L-[35S]cysteine into tRNA that was bound on the discs, and the rates were assessed in the linear range of the progress curve at Ͻ10% of the product formation

Summary

Introduction

All organisms chemically modify the bases of their tRNA [1, 2]. The purpose of these modified nucleosides is varied. Because each modification is enzymatically introduced at the post-transcriptional level, the modification enzymes must recognize a specific base within a highly structured tRNA molecule. These systems provide another context for the study of protein-RNA interactions. The mechanism involves mobilization of sulfur from cysteine by IscS in the form of an enzyme-bound persulfide [23, 24], which is transferred to a cysteine residue on ThiI before final insertion into U8 of tRNA [25,26,27]. ThiI has the multiple roles of binding tRNA, activating O4 of uridine 8 using Mg-ATP while accepting a sulfur atom from IscS and transferring it to the 4-position to give the final product, s4U. To gain insight on how ThiI recognizes its tRNA substrate, we report on the substrate specificity for s4U modification using variants of unmodified E. coli tRNAs

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