title: Mechanism of action of RNase T. I. Identification of residues required for catalysis, substrate binding, and dimerization.
abstract: Escherichia coli RNase T, an RNA-processing enzyme and a member of the DEDD exonuclease superfamily, was examined using sequence analysis and site-directed mutagenesis. Like other DEDD exonucleases, RNase T was found to contain three conserved Exo motifs that included four invariant acidic residues. Mutagenesis of these motifs revealed that they are essential for RNase T activity, indicating that they probably form the RNase T catalytic center in a manner similar to that found in other DEDD exonucleases. We also identified by sequence analysis three short, but highly conserved, sequence segments rich in positively charged residues. Site-directed mutagenesis of these regions indicated that they are involved in substrate binding. Additional analysis revealed that residues within the C-terminal region of RNase T are essential for RNase T dimerization and, consequently, for RNase T activity. These data define the domains necessary for RNase T action, and together with information in the accompanying article, have led to the formulation of a detailed model for the structure and mechanism of action of RNase T.
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D002384:Catalysis; D002850:Chromatography, Gel; D019281:Dimerization; D004926:Escherichia coli / Q000201:enzymology*; D005095:Exoribonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012343:RNA, Transfer / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013696:Temperature
chemical_list: D012343:RNA, Transfer; D005095:Exoribonucleases; C042081:exoribonuclease T