ABSTRACT
To date the only tRNAs containing nucleosides modified with a selenium (5-carboxymethylaminomethyl-2-selenouridine and 5-methylaminomethyl-2-selenouridine) have been found in bacteria. By using tRNA anticodon-stem-loop fragments containing S2U, Se2U, or geS2U, we found that in vitro tRNA 2-selenouridine synthase (SelU) converts S2U-RNA to Se2U-RNA in a two-step process involving S2U-RNA geranylation (with ppGe) and subsequent selenation of the resulting geS2U-RNA (with SePO33- ). No 'direct' S2U-RNAâSe2U-RNA replacement is observed in the presence of SelU/SePO33- only (without ppGe). These results suggest that the in vivo S2UâSe2U and S2UâgeS2U transformations in tRNA, so far claimed to be the elementary reactions occurring independently in the same domain of the SelU enzyme, should be considered a combination of two consecutive events - geranylation (S2UâgeS2U) and selenation (geS2UâSe2U).
Subject(s)
Escherichia coli/enzymology , Organoselenium Compounds/metabolism , Selenium/metabolism , Sulfurtransferases/physiology , Terpenes/metabolism , Uridine/analogs & derivatives , Binding Sites , Carbon/metabolism , Catalysis , Escherichia coli/genetics , Polyisoprenyl Phosphates/metabolism , Protein Processing, Post-Translational/genetics , RNA, Bacterial/chemistry , RNA, Bacterial/metabolism , RNA, Transfer/chemistry , RNA, Transfer/metabolism , Sulfurtransferases/genetics , Thiouridine/chemistry , Thiouridine/metabolism , Uridine/metabolismABSTRACT
Recently, highly lipophilic S-geranylated derivatives of 5-methylaminomethyl-2-thiouridine (mnm5geS2U) and 5-carboxymethylaminomethyl-2-thiouridine (cmnm5geS2U) were found at the first (wobble) anticodon position in bacterial tRNAs specific for Lys, Glu and Gln. The function and cellular biogenesis of these unique tRNAs remain poorly understood. Here, we present one direct and two post-synthetic chemical routes for preparing model geS2U-RNAs. Our experimental data demonstrate that geS2U-RNAs are more lipophilic than their parent S2U-RNAs as well as non-modified U-RNAs. Thermodynamic studies revealed that the S-geranyl-2-thiouridine-containing RNA has higher affinity toward complementary RNA strand with G opposite the modified unit than with A. Recombinant tRNA selenouridine synthase (SelU) exhibits sulfur-specific geranylation activity toward model S2U-RNA, which is composed of the anticodon-stem-loop (ASL) from the human tRNALys3 sequence. In addition, the presence of magnesium ions is required to achieve appreciable geranylation efficiencies.