Abstract
The wobble nucleoside 5-methylaminomethyl-2-thio-uridine (mnm5s2U) is present in bacterial tRNAs specific for Lys and Glu and 5-carboxymethylaminomethyl-2-thio-uridine (cmnm5s2U) in tRNA specific for Gln. The sulfur of (c)mnm5s2U may be exchanged by selenium (Se)–a reaction catalyzed by the selenophosphate-dependent tRNA 2-selenouridine synthase encoded by the mnmH (ybbB, selU, sufY) gene. The MnmH protein has a rhodanese domain containing one catalytic Cys (C97) and a P-loop domain containing a Walker A motif, which is a potential nucleotide binding site. We have earlier isolated a mutant of Salmonella enterica, serovar Typhimurium with an alteration in the rhodanese domain of the MnmH protein (G67E) mediating the formation of modified nucleosides having a geranyl (ge)-group (C10H17-fragment) attached to the s2 group of mnm5s2U and of cmnm5s2U in tRNA. To further characterize the structural requirements to increase the geranylation activity, we here report the analysis of 39 independently isolated mutants catalyzing the formation of mnm5ges2U. All these mutants have amino acid substitutions in the rhodanese domain demonstrating that this domain is pivotal to increase the geranylation activity. The wild type form of MnmH+ also possesses geranyltransferase activity in vitro although only a small amount of the geranyl derivatives of (c)mnm5s2U is detected in vivo. The selenation activity in vivo has an absolute requirement for the catalytic Cys97 in the rhodanese domain whereas the geranylation activity does not. Clearly, MnmH has two distinct enzymatic activities for which the rhodanese domain is pivotal. An intact Walker motif in the P-loop domain is required for the geranylation activity implying that it is the binding site for geranylpyrophosphate (GePP), which is the donor molecule in vitro in the geranyltransfer reaction. Purified MnmH from wild type and from the MnmH(G67E) mutant have bound tRNA, which is enriched with geranylated tRNA. This in conjunction with earlier published data, suggests that this bound geranylated tRNA may be an intermediate in the selenation of the tRNA.
Highlights
Transfer RNA plays a pivotal role in translating the information residing in mRNA into proteins
The first alteration in MnmH was discovered in a mutant mediating the ability to suppress +1 frameshift mutation and this induced phenotype is due to geranylation of tRNAGcmlnnm5s2UUG [4] alteration is within the 36 amino acid insertion in the rhodanese domain conserved among bacterial and archaeal MnmH orthologues (Fig 2)
The alterations were in the vicinity of this 36 amino acid insertion and all were close to the region where the catalytic Cys97 of the rhodanese domain is located
Summary
Transfer RNA plays a pivotal role in translating the information residing in mRNA into proteins. Position 34 (the wobble nucleoside) and position 37 (the nucleoside adjacent and 3 ́of the anticodon) in tRNAs from all organisms are frequently modified (61% and 75%, respectively) but a large variety of different modified nucleosides are present in these two positions [1]C:\GetARef\Refs\CTS_2012.ref #1;. These modified nucleosides in tRNA are important for a proper and efficient reading of the mRNA and lack of some of them results in severe physiological defects and/or no viability (Reviewed in [1]). Several of these modified nucleosides have different chemical structures, are present in different tRNAs, and at different positions in the tRNA, many of them are important for keeping the ribosome in the correct reading frame [2]
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