Mechanism of action of RNase T. II. A structural and functional model of the enzyme.
A detailed structural and functional model of E. coli RNase T was generated based on sequence analysis, homology modeling, and experimental observation. In the accompanying article, three short sequence segments (nucleic acid binding sequences (NBS)) important for RNase T substrate binding were identified. In the model, these segments cluster to form a positively charged surface patch. However, this patch is on the face of the RNase T monomer opposite the DEDD catalytic center. We propose that by dimerization, the NBS patch from one subunit is brought to the vicinity of the DEDD center of the second monomer to form a fully functional RNase T active site. In support of this model, mutagenetic studies show that one NBS1 residue, Arg(13), sits at the catalytic center despite being on the opposite side of the monomer. Second, the complementarity of the RNase T subunits through the formation of homodimers was demonstrated by reconstitution of partial RNase T activity from monomers derived from two inactive mutant proteins, one defective in catalysis and one in substrate binding. These data explain why RNase T must dimerize to function. The model provides a detailed framework on which to explain the mechanism of action of RNase T.
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001665:Binding Sites; D002384:Catalysis; D019281:Dimerization; D004926:Escherichia coli / Q000201:enzymology*; D005095:Exoribonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; 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; D012984:Software; D013329:Structure-Activity Relationship; D013696:Temperature
D012343:RNA, Transfer; D005095:Exoribonucleases; C042081:exoribonuclease T