Abbreviated Pathway for Biosynthesis of 2-Thiouridine in Bacillus subtilis

Abstract

The 2-thiouridine (s 2 U) modification of the wobble position in glutamate, glutamine, and lysine tRNA molecules serves to stabilize the anticodon structure, improving ribosomal binding and overall efficiency of the translational process. Biosynthesis IMPORTANCEThe 2-thiouridine (s 2 U) modification of the wobble position in glutamate, glutamine, and lysine tRNA is conserved in all three domains of life and stabilizes the anticodon structure, thus guaranteeing fidelity in translation. The biosynthesis of s 2 U in Escherichia coli requires seven proteins: the cysteine desulfurase IscS, the thiouridylase MnmA, and five intermediate sulfur-relay enzymes (TusABCDE). Bacillus subtilis and most Gram-positive bacteria lack a complete set of biosynthetic components. Interestingly, the mnmA coding sequence is located adjacent to yrvO, encoding a cysteine desulfurase. In this work, we provide evidence that the B. subtilis YrvO is able to transfer sulfur directly to MnmA. Both proteins are sufficient for s 2 U biosynthesis in a pathway independent of the one used in E. coli. P osttranscriptional RNA modifications are found among all organisms and are essential for their cellular function. More than 100 RNA modifications have been identified thus far which have a variety of physiological functions (1). In particular, posttranscriptional modifications of tRNA are often necessary for translational fidelity and efficiency, especially those found in anticodon bases. The third base in the anticodon is deemed the wobble position, as it is bound more loosely to the ribosome than the first two. Consequently, wobble bases have greater flexibility, allowing them to make noncanonical base pairs, leading to a greater chance of amino acid misincorporation into a growing peptide chain. An explanation of how this phenomenon is overcome is provided by the modified wobble hypothesis, which states that certain base modifications have evolved to adjust the anticodon shape, either to limit or to enable the occurrence of wobble pairing (2).The modification of the wobble (34th position) in glutamate (Glu), glutamine (Gln), and lysine (Lys) tRNA molecules produces 5-methyl-2-thiouridine derivatives (xm 5 s 2 U) (Fig. 1). Thiolation of the wobble uridine places the base in an anti, C-3=-endo, gauche conformation, thus forming a rigid structure, which increases its affinity for pairing with adenosine and thereby yields higher accuracy in the corresponding peptide sequence. Additionally, this modification stabilizes the anticodon structures and confers the tRNA molecule's ability to bind to the ribosome. Tighter ribosomal binding to tRNA subsequently improves reading frame maintenance and deficiencies in the translational process (2-5). Lack of the 2-thiouridine (s 2 U) modification in tRNA causes growth defects in bacteria attributed to accumulation of frameshifting during translation (6). In humans, mutations within the gene coding for the 2-thiouridylase TrmU, responsible for the sulfur insertion step, has been associated with acute in...

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

“…The Bacillus subtilis ThiI appears to be a dedicated protein in s 4 U biosynthesis and it is not required for thiamine generation [165]. Interestingly, B. subtilis contains four cysteine desulfurases and evidence thus far has demonstrated that each pairs with a dedicated sulfur transferase for specific thiocofactor generation [68,72,166]. Formation of s 4 U, in particular, is accomplished by the cysteine desulfurase NifZ, the coding sequence of which is found immediately upstream of thiI .…”

“…Interestingly, in B. subtilis and in several other Gram-positive species missing tus genes, an IscS-like cysteine desulfurase gene ( yrvO ) is located in the same genomic region, and commonly directly upstream, of mnmA . Experimental characterization of B. subtilis YrvO and MnmA both in vivo and in vitro demonstrated that these two proteins are the sole requirements for s 2 U formation in this bacterium [72]. It is worth noting that the B. subtilis genome encodes a rhodanese protein (YrkF) with homology to TusA which is a competent sulfur acceptor of YrvO, but inactivation of yrkF leads to no changes in s 2 U levels [72,174].…”

Biosynthesis of Sulfur-Containing tRNA Modifications: A Comparison of Bacterial, Archaeal, and Eukaryotic Pathways

“…Notably, the rhodanese homology domain (RHD) that contains the catalytic Cys456, which carries the persulfide, is absent in many bacteria [8,45]; therefore, the sulfur transfer mechanism of RHD-lacking ThiI remains unanswered. The genome of B. subtilis encodes four functionally active cysteine desulfurases: SufS, NifZ, NifS, and YrvO [50]. Among them, the nifZ gene is adjacent to the thiI gene without RHD.…”

“…Recently, a truncated pathway of s 2 U34 biosynthesis has been revealed in B. subtilis [50]. In this bacterium, the intermediate sulfur carries (TusA/BCD/E) are missing (Figure 4B).…”

“…In this bacterium, the intermediate sulfur carries (TusA/BCD/E) are missing (Figure 4B). The cysteine desulfurase YrvO and MnmA are sufficient to introduce s 2 to U34 [50]; this suggests a direct sulfur transfer from a cysteine desulfurase to the s 2 U34 formation enzyme.…”