Characterization of Escherichia coli RNase PH.
We have previously shown that the orfE gene of Escherichia coli encodes RNase PH. Here we show that the OrfE protein (purified as described in the accompanying paper) (Jensen, K. F., Andersen, J. T., and Poulsen, P. (1992) J. Biol. Chem. 267, 17147-17152) has both the degradative and synthetic activities of RNase PH. This highly purified protein was used to characterize the enzymatic and structural properties of RNase PH. The enzyme requires a divalent cation and phosphate for activity, the latter property indicating that RNase PH is exclusively a phosphorolytic enzyme. Among tRNA-type substrates, the enzyme is most active against synthetic tRNA precursors containing extra residues following the -CCA sequence, and it can act on these molecules to generate mature tRNA with amino acid acceptor activity; 3'-phosphoryl-terminated molecules are not active as substrates. The equilibrium constant for RNase PH is near unity, suggesting that at the phosphate concentration present in vivo, the enzyme would participate in RNA degradation. The synthetic reaction of RNase PH displays a nonlinear response to increasing enzyme concentrations, and this may be due to self-aggregation of the protein. Higher order multimers of RNase PH could be detected by gel filtration at higher protein concentrations and by protein cross-linking. The possible role of RNase PH in tRNA processing is discussed.
Kelly
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Department of Biochemistry, University of Connecticut Health Center, Farmington 06030.
Deutscher
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D001483:Base Sequence; D002413:Cations, Divalent; D002850:Chromatography, Gel; D004120:Dimethyl Suberimidate / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005095:Exoribonucleases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D005798:Genes, Bacterial; D007700:Kinetics; D012322:RNA Precursors / Q000378:metabolism; D012343:RNA, Transfer / Q000378:metabolism*; D013379:Substrate Specificity
D002413:Cations, Divalent; D012322:RNA Precursors; D004120:Dimethyl Suberimidate; D012343:RNA, Transfer; C056502:ribonuclease PH; D005095:Exoribonucleases