Characterization of the biochemical properties of recombinant ribonuclease III.
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.
Department of Biochemistry, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway 08854.
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March
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Gonzalez
D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D004722:Endoribonucleases / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D029968:Escherichia coli Proteins; D019204:GTP-Binding Proteins / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D011189:Potassium Chloride; D012329:RNA, Bacterial / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D043244:Ribonuclease III; D012996:Solutions
D003432:Cross-Linking Reagents; D029968:Escherichia coli Proteins; D012329:RNA, Bacterial; D011994:Recombinant Proteins; D012996:Solutions; D011189:Potassium Chloride; D004722:Endoribonucleases; D043244:Ribonuclease III; C468366:ribonuclease III, E coli; D019204:GTP-Binding Proteins