Sulfur-deficient transfer ribonucleic acid. Natural substrate for ribonucleic acid sulfurtransferase from Escherichia coli

Abstract

Sulfur-deficient tRNA, isolated after cysteine starvation of Escherichia coli HfrC, RCre', met-, cys-, A, was used as the substrate for the crude mixture of E. coli RNA sulfurtransferases. This tRNA accepted nearly four times more sulfur in vitro than normal tRNA. Sulfur incorporation was inversely proportional to the 4-thiouridine content. The optimal concentrations of all of the required cofactors and substrates, ATP, Mg2+, cysteine, and tRNA were determined. The apparent K , for cysteine was 2.6 X M and that for sulfur-deficient tRNA, 2.7 x M. Chromatography of

Mammalian tRNA sulfurtransferase: properties of the enzyme in rat liver

“…Sulfurdeficient tRNA was isolated from E. coli C6 as previously described (24). normal thionucleotide content (20). These data further suggest that the sites available for thiolation with liver enzymes are different than those recognized by the E. coli enzymes.…”

“…Only (11,14,20) and rat brain (15) also contain thionucleotides which chromatograph at this position on DEAE-cellulose. We are currently evaluating the isolated hepatocyte system (25) and certain cell lines maintained in culture to see if [35 ] tRNA can be isolated for use as an in vivo marker, and an aid to identification of the presumed in vitro labeled thionucleotide.…”

“…Our previous experiments were carried out at pH 8.5 (17), the optimal pH of the bacterial system (12,20). This would cause a reduction in activity of about 20% for the assays carried out at the higher pH.…”

Increased isoleucine acceptance by sulfur-deficient transfer RNA from Escherichia coli

“…Initial rates were determined at low synthetase levels and before 10 per cent of the reaction was completed. d) Preparation and Assay of RNA Sulfurtransferases A crude mixture of RNA sulfurtransferases was prepared from E. coli Q13 by a modification of the procedure of Hayward and Weiss (16), and has been previously described (11). The enzyme preparation was stable at -20°f or several weeks.…”

“…We previously reported a marked increase in isoleucine acceptance for sulfur-deficient tRNA, isolated after starvation of a cysteine-requiring, RCral strain of E. coli (10). This tRNA is a mixture of thiolated and unthiolated tRNA, the latter fraction being deficient in all of the known thionucleotides (4,11). Sulfur-deficient tRNA can be rethiolated l1 vitro with a mixture of Uk eulfurtransferases, thus affording the study of tRNA function with and without thionucleotides.…”