Substrate Specificity for 4-Thiouridine Modification in Escherichia coli

Abstract

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 T⌿C 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 s 4 U modification is limited in the cell to tRNA.

Deduced RNA binding mechanism of ThiI based on structural and binding analyses of a minimal RNA ligand

“…Taken together, these results indicate that the acceptor-stem region plays an important role in binding with ThiI, which is in good agreement with a previous report by Lauhon et al (2004), indicating that deleting nucleotides in the acceptor-stem and/or 39-terminal nucleotides markedly impaired modification activity. On the other hand, Lauhon et al (2004) also reported that replacing sequences of the T-loop by a GAAA sequence in truncated tRNA significantly increased the activity. In TPHE39A, the T-stem was disrupted and formed a large bulged loop together with the T-loop sequences (Fig.…”

“…CD spectra showed that no significant structural change occurred in TPHE39A on binding with ThiI, which allowed us to discuss the binding mechanism between ThiI and TPHE39A based on the determined structure of TPHE39A. Taken together, these results indicate that the acceptor-stem region plays an important role in binding with ThiI, which is in good agreement with a previous report by Lauhon et al (2004), indicating that deleting nucleotides in the acceptor-stem and/or 39-terminal nucleotides markedly impaired modification activity. On the other hand, Lauhon et al (2004) also reported that replacing sequences of the T-loop by a GAAA sequence in truncated tRNA significantly increased the activity.…”

“…The dissociation constants of ThiI against tRNA Phe and TPHE39A calculated from association constants determined by ITC were 0.25 6 0.06 and 0.56 6 0.14 mM, respectively, which were markedly smaller than those previously reported using a filter binding assay (1.9 6 0.17 and 8.2 6 1.7 mM, respectively) ( Table 2; Lauhon et al 2004). This would be due to the difference of the methods used.…”

“…Among these, previous mutational analysis showed that Cys344 and Cys456 are important for the activity (Palenchar et al 2000;Mueller et al 2001). Lauhon et al (2004) examined the substrate specificity for s 4 U modification by deletion analysis of tRNA Phe , and showed that TPHE39A, consisting of 39 nucleotides (nt) including the acceptor-stem, a bulged loop, the T-stem, and the modified T-loop, was the minimal efficient substrate with activity nearly two-thirds that of full-length tRNA Phe , i.e., initial rate constant (k obs ) and dissociation constant (K d ) of tRNA Phe and TPHE39A were 1.01 6 0.13 min À1 and 1.9 6 0.17 mM and 0.61 6 0.06 min À1 and 8.2 6 1.7 mM, respectively. It was also reported that: (1) the bulged loop must be at least 4 nt in length, and contain the target uridine as the first nucleotide; (2) the primary sequence in the T-loop is not a recognition element; and (3) a 39-CCA overhang is required for activity.…”

Biosynthesis of 4-Thiouridine in tRNA in the Methanogenic Archaeon Methanococcus maripaludis

“…The K m for Na 2 S was 1.0 Ϯ 0.2 mM, which was within the range of intracellular free sulfide concentration in methanococci of 1-3 mM (59). This result suggests that MMP1354 has a much higher affinity for sulfide than E. coli ThiI (K m Ͼ20 mM) (26), and sulfide is a physiologically relevant sulfur donor for s 4 U biosynthesis in M. maripaludis. Furthermore, unlike the E. coli ThiI that requires exogenous reductant for multiple turnovers (32), the addition of DTT inhibited the s 4 U formation by MMP1354 (Fig.…”