Crystal structure of a 4-thiouridine synthetase–RNA complex reveals specificity of tRNA U8 modification

Abstract

In prokaryotes and archaea transfer ribonucleic acid (tRNA) stability as well as cellular UV protection relies on the post-transcriptional modification of uracil at position 8 (U8) of tRNAs by the 4-thiouridine synthetase ThiI. Here, we report three crystal structures of ThiI from Thermotoga maritima in complex with a truncated tRNA. The RNA is mainly bound by the N-terminal ferredoxin-like domain (NFLD) and the THUMP domain of one subunit within the ThiI homo-dimer thereby positioning the U8 close to the catalytic center in the pyrophosphatase domain of the other subunit. The recognition of the 3’-CCA end by the THUMP domain yields a molecular ruler defining the specificity for U8 thiolation. This first structure of a THUMP/NFLD-RNA complex might serve as paradigm for the RNA recognition by THUMP domains of other proteins. The ternary ThiI–RNA–ATP complex shows no significant structural changes due to adenosine triphosphate (ATP) binding, but two different states of active site loops are observed independent of the nucleotide loading state. Thereby conformational changes of the active site are coupled with conformational changes of the bound RNA. The ThiI–RNA complex structures indicate that full-length tRNA has to adopt a non-canonical conformation upon binding to ThiI.

Trm112, a Protein Activator of Methyltransferases Modifying Actors of the Eukaryotic Translational Apparatus

“…Such modular organization has been confirmed by the recent crystal structure of the archaeal Trm11 ortholog from Thermococcus kodakarensis [28] and is shared with archaeal Trm14 and bacterial TrmN, which are both responsible for m 2 G formation at position 6 on tRNAs [29,30]. In the 4-thiouridine synthase enzyme ThiI, the THUMP domain was shown to interact with the 3’ CCA end [31] and was then proposed to position the substrate nucleotide in the enzyme active site. It would then act as a molecular ruler that controls the distance between the tRNA CCA end and the nucleotide to be modified.…”

Diverse Mechanisms of Sulfur Decoration in Bacterial tRNA and Their Cellular Functions

“…While the C-terminal rhodanese sulfur transfer domain appears to be missing in certain ThiI sequences, structural and sequence analyses reveal the presence of three additional majorly conserved domains [67,69] comprehending a large thiouridylase domain. The THUMP (named after thiouridine synthases, RNA methylases and pseudouridine synthases) domain is involved in recognition of the 3′-CCA end of tRNA and positioning the uridine in the correct orientation for catalysis [67].…”

“…While the C-terminal rhodanese sulfur transfer domain appears to be missing in certain ThiI sequences, structural and sequence analyses reveal the presence of three additional majorly conserved domains [67,69] comprehending a large thiouridylase domain. The THUMP (named after thiouridine synthases, RNA methylases and pseudouridine synthases) domain is involved in recognition of the 3′-CCA end of tRNA and positioning the uridine in the correct orientation for catalysis [67]. The N-terminal Ferredoxin-like domain (NFLD) is also involved in the binding of tRNA [69], while the C-terminal PPase domain contains an adenylation-specific PP-loop motif (SGGFDS) belonging to the PP i synthetase superfamily [65].…”

The Evolution of Substrate Specificity by tRNA Modification Enzymes

“…The crystal structure of the Thermotoga maritima Thil 4-thiouridine synthase, in complex with a truncated tRNA, confirmed the role of THUMP domains in RNA binding and revealed a potential molecular ruler mechanism to define substrate specificity [58]. Interestingly, like Thil, the RNA methyltransferases Trm11, RlmKL, Trm14/TrmN, and RlmN (a radical SAM enzyme) all contain an N-terminal ferredoxin-like (NFLD) domain along with the THUMP domain, implying that THUMP and NFLD together could form a functional binding unit as is seen in Thil [58]. For example, Trm11 from Eukarya and Archaea is comprised of three domains: NFLD, THUMP, and a class I methyltransferase catalytic domain (Fig.…”