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.
Highlights
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
RNase T is an important enzyme for various aspects of RNA metabolism, it was found to act on singlestranded DNA substrates in vitro (9, 10)
Summary
Additional analysis revealed that residues within the C-terminal region of RNase T are essential for RNase T dimerization and, 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. RNase T is one of eight exoribonucleases present in Escherichia coli (1) It belongs to the DEDD exonuclease superfamily, characterized by common motifs containing four invariant acidic residues, which in DNA polymerases were shown to form the exonuclease active site (2). We have shown that the C-terminal region of RNase T is important for RNase T dimerization and, for activity These data identify residues needed for RNase T action and provide essential information for the development of a detailed model of RNase T action presented in the accompanying article (15)
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