Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.26.3 (RNase III)
 1,015 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

An Escherichia coli double strand specific endoribonuclease, RNase III, was cloned, expressed in large amounts, and purified to homogeneity. Enzyme activity was monitored by assaying fractions for the ability to correctly process exogenous RNA containing specific RNase III cleavage sites. DEAE-Sepharose ion exchange chromatography in the presence of a linear KCl gradient (from 0.02 M to 0.75 M) demonstrated that RNase III exists as two distinct forms. One form elutes at a KCl concentration of 0.13 M and the other elutes at 0.33 M. The presence of stoichiometric amounts of the GTP-binding protein Era during purification results in the conversion of the low salt form into the high salt form. Size exclusion chromatography demonstrated that both forms exist as a dimer in solution. In order to investigate the nature of the dimer, protein cross-linking was performed and cross-linked products were detected by silver staining. The protein-protein dimer can be visualized at protein:cross-linker molar ratios as low as 1:15 within 1 minute of exposure to cross-linker in 0.1 M KCl. Upon addition of substrate RNA to the cross-linking reaction a second form of the protein-protein dimer (with a slightly smaller apparent molecular weight) becomes prominent. Induction of the new form is absolutely dependent upon the addition of substrate mRNA to the reaction mixture. We postulate that the RNase III dimer undergoes a dramatic conformational change upon recognition of RNA which we are able to trap by cross-linking.
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PMID:Characterization of the biochemical properties of recombinant ribonuclease III. 169 24

Using RNA-directed synthesis of the alpha-peptide of beta-galactosidase as an assay, a factor was purified that inactivated further function of the mRNA. In the presence of Ca2+ ions to inhibit most nuclease activity, inactivation of mRNA occurred during incubation with ribosomes or with a 1 M KCl wash of ribosomes. The inactivation activity required Mg2+ ions, and purified as a single factor which did not bind to DEAE-cellulose, but bound reversibly to phosphocellulose. The factor eluted from Sephadex G-150 with an apparent molecular weight of about 43,000. Purified 700-fold, it showed no detectable exonuclease activity, and little or no cleavage of a variety of single-stranded substrates, including full length lac operon mRNA; but repurified inactivated mRNA was still inactive for protein synthesis. The factor did not inhibit poly(U)-directed polyphenylalanine synthesis. When proteins isolated from the ribosomal wash were individually tested, highly purified RNase III, which purifies in the same way and has the same size, also inactivated lac mRNA. The ribosomal wash from an RNase III- strain showed little if any activity compared to that from an isogenic RNase III+ strain. The possibility of a site-specific inactivating cleavage of mRNA by RNase III at or near the 5' end is considered.
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PMID:Functional inactivation of lac alpha-peptide mRNA by a factor that purifies that Escherichia coli RNase III. 625 91