Substrate specificity for 4-thiouridine modification in Escherichia coli

The biosynthesis of 4-thiouridine (s(4)U) 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 s(4)U. In this work, we show through deletion analysis of unmodified E. coli tRNA(Phe) that the minimum substrate for s(4)U 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 tRNAPhe 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 s(4)U modification is limited in the cell to tRNA.