Abstract
RNase T, a nuclease thought to be involved in end-turnover of tRNA, has been purified about 4,000-fold from extracts of Escherichia coli. At this stage of purification, the enzyme was judged to be at least 95% pure based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The native molecular weight of RNase T determined from gel filtration and sedimentation analyses is about 50,000, whereas the monomer molecular weight determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is 25,000, suggesting that the protein is an alpha 2 dimer. Purified RNase T is extremely sensitive to inactivation by oxidation, sulfhydryl group reagents, and temperature. The ribonuclease activity against tRNA-C-C-[14C]A is optimal at pH 8-9 in the presence of 2-5 mM MgCl2 and ionic strengths of less than 50mM. Although RNase T is highly specific for intact tRNA-C-C-A as a substrate and can hydrolyze all species in a mixed population of tRNA, it is inhibited by other RNAs, such as poly(A), rRNA, 5 S RNA, and tRNA-C-C. RNase T is an exoribonuclease which initiates attack at a free 3' terminus of tRNA and releases AMP; aminoacyl-tRNA is not a substrate. The role of RNase T in the end-turnover of tRNA and its possible involvement in other aspects of RNA metabolism are discussed.
Highlights
Turnover of tRNA, has been purified about 4,000-fold Strains andGrowth Conditions-E. coli K12 strain C3/5-7
The strainwas rification, the enzyme was judged to be at least 95% derived from thepreviously described C3/5(8) by P1-mediated transpure based on sodium dodecyl sulfate-polyacrylamide duction to introduce the mutant rnlobcus, using cotransduction with gel electrophoresis
RNase T determined from gel filtration and sedimentation analyses is about 50,000, whereas the monomer molecular weight determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is 25,000,suggesting that the protein is an a2dimer
Summary
Turnover of tRNA, has been purified about 4,000-fold Strains andGrowth Conditions-E. coli K12 strain C3/5-7 This procedure leads to an apparent purification of RNase T from a high-speed supernatant fraction of about dithiothreitol, we repeatedly observed loss of enzyme activity which could be reversed almost completely by incubation with the reducing agent. Purity-The coincident elution of RNase T activity and protein (based on A220) from the Ultrogel AcA44 column suggested thatthe enzymewashighly purified. This was demonstrated by SDS-PAGE of the combined, concentrated fraction (Fig. 2 A ) whichshowed a single band migrating slightly behind the trypsinogen standard. When detection of protein after SDS-PAGE was carried out with the more sensitive silver staining procedure, a minor band of slightly higher molecular weight was observed (Fig. 2B) This contaminant could be removed bya second chromatographic step on hydroxylapatite.'.
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