Functional Diversity of the Rhodanese Homology Domain

2-Thioribothymidine (s 2 T) is a modified nucleoside of U, specifically found at position 54 of tRNAs from extreme thermophilic microorganisms. The function of the 2-thiocarbonyl group of s 2 T54 is thermostabilization of the three-dimensional structure of tRNA; however, its biosynthesis has not been clarified until now. Using an in vivo tRNA labeling experiment, we demonstrate that the sulfur atom of s 2 T in tRNA is derived from cysteine or sulfate. We attempted to reconstitute 2-thiolation of s 2 T in vitro, using a cell extract of Thermus thermophilus. Specific 2-thiolation of ribothymidine, at position 54, was observed in vitro, in the presence of ATP. Using this assay, we found a strong temperature dependence of the 2-thiolation reaction in vitro as well as expression of 2-thiolation enzymes in vivo. These results suggest that the variable content of s 2 T in vivo at different temperatures may be explained by the above characteristics of the enzymes responsible for the 2-thiolation reaction. Furthermore, we found that another posttranscriptionally modified nucleoside, 1-methyladenosine at position 58, is required for the efficient 2-thiolation of ribothymidine 54 both in vivo and in vitro.Post-transcriptional modification is a characteristic feature of RNA molecules. In transfer RNA, post-transcriptional modification plays various roles that are required for the translation process, including fidelity control of codon recognition, reading frame maintenance, and stabilization of the tertiary tRNA structure (1).2-thioribothymidine (s 2 T) 2 is a 2-thiolated derivative of 5-methyluridine (ribothymidine (rT)), located at position 54 of Thermus thermophilus tRNA (2), and has been shown to stabilize tRNA structure in a high temperature environment (3). In the extreme thermophilic eubacteria, Thermus thermophilus (4), and the archaea, Pyrococcus furiosus (5), the 2-thiolation level of rT54 in tRNA increases with cultivation temperature; the melting temperature (T m ) of the tRNA increases concomitantly with incremental increases in s 2 T content. These findings indicate that 2-thiolation of rT54 is responsible for the thermostability of thermophile tRNA at a variety of cultivation temperatures, thereby ensuring the adaptation of the protein synthesis machinery to specific thermal environments.However, the body of knowledge regarding s 2 T biosynthesis is limited. First, the enzymes responsible for the tRNA modification and the genes involved in the 2-thiolation reaction have not been identified to date. Previously, we demonstrated that the tRNA structural elements required for 2-thiolation are the conserved bases in the TC loop and the structure created by those bases (6). In this report, we investigated the sulfur donor for s 2 T biosynthesis in vivo. We also examined the 2-thiolation reaction in vitro, in order to characterize the temperature dependence of this reaction, and considered the contribution of neighboring nucleoside modifications to the efficiency of 2-thiolation of rT54, both in vivo and in vitro. ...

supporting

confidence: 58%

mentioning

confidence: 99%

Temperature-dependent Biosynthesis of 2-Thioribothymidine of Thermus thermophilus tRNA

Shigi

Suzuki

Terada

et al. 2006

“…The role of YnjE so far remained enigmatic for E. coli. In total, with the exception of ThiI and YbbB, which are required for thiamine/thiouridine and selenouridine biosynthesis (18,20), respectively, little is known about the in vivo role of the other rhodaneses. It was of interest to identify whether, by analogy to Moco biosynthesis in humans, a rhodanese-like protein acts as a mediator between IscS and MoaD for the formation of the thiocarboxylate group of MPT synthase in conjunction with MoeB.…”

mentioning

confidence: 99%

“…Furthermore, catalytic or inactive rhodanese domains are also found in other proteins, such as ThiI (16), or in Cdc25 phosphatases (17). In the E. coli genome, eight genes have been identified coding for proteins containing a rhodanese-like protein domain bearing a conserved cysteine residue as a potential active site for persulfide formation (15,18). Three of them code for the proteins GlpE, PspE, and YgaP that are composed of only a single rhodanese domain.…”

mentioning

confidence: 99%

The Identification of a Novel Protein Involved in Molybdenum Cofactor Biosynthesis in Escherichia coli

Dahl

Urban

Bolte

et al. 2011

Self Cite

Background: In Moco biosynthesis, sulfur is transferred from L-cysteine to MPT synthase, catalyzing the conversion of cPMP to MPT. Results: The rhodanese-like protein YnjE is a novel protein involved in Moco biosynthesis. Conclusion: YnjE enhances the rate of conversion of cPMP to MPT and interacts with MoeB and IscS. Significance: To understand the mechanism of sulfur transfer and the role of rhodaneses in the cell.

“…In tRNA mnm5s2UUU . 2 When selenium is available in the growth medium, sulfur at position 2 may be replaced by selenium in a reaction dependent on SelD and YbbB (17)(18)(19). It has been shown that modifications at position 2, but not at position 5, are important for aminoacylation of tRNAs (20)(21)(22), whereas modification in both 2-and 5-positions function in the codon recognition process (17,18,(22)(23)(24)(25)(26)(27).…”

mentioning

confidence: 99%

Effects of Mutagenesis in the Switch I Region and Conserved Arginines of Escherichia coli MnmE Protein, A GTPase Involved in tRNA Modification

Martínez-Vicente

Yim

Villarroya

et al. 2005

MnmE is an evolutionarily conserved, three domain GTPase involved in tRNA modification. In contrast to Ras proteins, MnmE exhibits a high intrinsic GTPase activity and requires GTP hydrolysis to be functionally active. Its G domain conserves the GTPase activity of the full protein, and thus, it should contain the catalytic residues responsible for this activity. In this work, mutational analysis of all conserved arginine residues of the MnmE G-domain indicates that MnmE, unlike other GTPases, does not use an arginine finger to drive catalysis. In addition, we show that residues in the G2 motif ( 249 GTTRD 253 ), which resides in the switch I region, are not important for GTP binding but play some role in stabilizing the transition state, specially Gly 249 and Thr 251 . On the other hand, G2 mutations leading to a minor loss of the GTPase activity result in a non-functional MnmE protein. This indicates that GTP hydrolysis is a required but non-sufficient condition so that MnmE can mediate modification of tRNA. The conformational change of the switch I region associated with GTP hydrolysis seems to be crucial for the function of MnmE, and the invariant threonine (Thr 251 ) of the G2 motif would be essential for such a change, because it cannot be substituted by serine. MnmE defects result in impaired growth, a condition that is exacerbated when defects in other genes involved in the decoding process are simultaneously present. This behavior is reminiscent to that found in yeast and stresses the importance of tRNA modification for gene expression.