Mechanism of Action of RNase T

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 dimeriza-tion 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. RNase T is one of eight exoribonucleases present in Esche-richia 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). RNase T plays an important role in stable RNA metabolism in E. coli, including tRNA end turnover (3) and 3 maturation of many stable RNAs (4-7). RNase T proteins are closely related to the proofreading domains/ subunits of bacterial DNA polymerases (2, 8), and, interestingly , E. coli RNase T also displays strong DNA exonuclease activity (9, 10). Although the substrates of RNase T in vivo share a common sequence feature consisting of a stable, double-stranded (ds) stem followed by a few unpaired 3 nucleotides, RNase T actually is a single-strand-specific exoribonuclease that acts in the 3-to-5 direction in a non-processive manner (11). However, whereas other E. coli exoribonucleases stop several nucleotides downstream of an RNA duplex, RNase T can digest RNA up to the first base pair, although it slows as it approaches the duplex structure. The presence of a free 3-hydroxyl group is required for the enzyme to initiate digestion (11). Homogeneous RNase T has been prepared from both normal and overexpressing cells (12, 13). The enzyme forms a ho-modimer in vitro and in vivo, and formation of the dimer seems to be required for it to function (14). Some residues needed for dimerization have been identified, as well as the importance of hydrophobicity in the dimerization process (13, 14). In this study, we investigate in detail structure-function relationships in RNase T using a combination of sequence analysis and site-directed mutagenesis. Our data indicate that the conserved acidic residues, as well as several other residues at the DEDD signature motifs, are important for RNase T catalytic activity, consistent with a common catalytic mechanism for all DEDD members. In addition, we also identified three other conserved sequence segments (the nucleic acid binding sequence (NBS) 1 segments), containing a high level of positively charged residues in RNase T orthologs. Kinetic analyses of the corresponding mutants suggest that the basic residues of the NBS segments are involved in nucleic acid-binding. Finally, we have shown that the C-terminal region of RNase T is important for RNase T dimerization and, consequently, 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). EXPERIMENTAL PROCEDURES Materials-Restriction endonucleases, T4 polynucleotide kinase, and T4 DNA ligase were obtained from New England Biolabs. Calf intestine alkaline phosphatase was purchased from Promega. [-32 P]ATP (6000 Ci/mmol) was from PerkinElmer Life Sciences. Sequagel, for single nucleotide resolution analysis, was purchased from National Diagnos-tics. The QIAEX II gel extraction kit was purchased from Qiagen Inc. The RNase T substrate, tRNA-CC -[ 3 H]A, was prepared by [ 3 H]A incorporation into tRNA-CC using tRNA nucleotidyltransferase and [ 3 H]ATP as described previously (12). DNA oligonucleotides were synthesized by the DNA Core Facility of our department. Ultrogel AcA44 was from Amersham Biosciences. The gel filtration calibration kit was from Pharmacia. Peroxidase-labeled anti-rabbit IgG and the ECL sub-strate were from Amersham Biosciences. All other chemicals were reagent grade. RNase T Activity Assay-RNase T reactions were carried out under buffer conditions containing 20 mM Tris-HCl, pH 8.0, 10 mM MgCl 2 , 50 mM KCl, and 5 mM dithiothreitol. Sonicated cell extracts were prepared as described previously (16) and were used for all activity measurements. Reaction mixtures were incubated at the indicated temperature for 5 min. For tRNA substrates, acid-soluble radioactivity was determined (12), whereas when the tetranucleotide, dA4, and the dinucle-otide, dA2, were used as substrates, reactions were stopped with 2 volumes of loading buffer containing 96% formamide and 1 mM EDTA, and the reaction products were analyzed by denaturing polyacrylamide gel electrophoresis on a 22.5% gel. Quantitative data were obtained