PMID:10625642
| Citation |
Callahan, C, Neri-Cortes, D and Deutscher, MP (2000) Purification and characterization of the tRNA-processing enzyme RNase BN. J. Biol. Chem. 275:1030-4 |
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| Abstract |
RNase BN, a tRNA-processing enzyme previously shown to be required for the 3'-maturation of certain bacteriophage T4-encoded tRNAs, was overexpressed and purified to near homogeneity from Escherichia coli. The purified enzyme, which is free of nucleic acid, is an alpha(2)-dimer with a molecular mass of approximately 65 kDa. RNase BN displays a number of unusual catalytic properties compared with the other exoribonucleases of E. coli. The enzyme is most active at pH 6.5 in the presence of Co(2+) and high concentrations of monovalent salts. It is highly specific for tRNA substrates containing an incorrect residue within the universal 3'-CCA sequence. Thus, tRNA-CU and tRNA-CA are effective substrates, whereas intact tRNA-CCA, elongated tRNA-CCA-Cn, phosphodiesterase-treated tRNA, and the closely related tRNA-CC are essentially inactive as substrates. RNA or DNA oligonucleotides also are not substrates. These data indicate that RNase BN has an extremely narrow substrate specificity. However, since tRNA molecules with incorrect residues within the -CCA sequence are not normally produced in E. coli, the role of RNase BN in uninfected cells remains to be determined. |
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| Keywords |
Base Sequence; Chromatography, Affinity; Chromatography, Gel; Chromatography, Ion Exchange; Cobalt/pharmacology; Dimerization; Enzyme Stability; Escherichia coli/enzymology; Exoribonucleases/chemistry; Exoribonucleases/isolation & purification; Exoribonucleases/metabolism; Hot Temperature; Hydrogen-Ion Concentration; Kinetics; Magnesium/pharmacology; Molecular Weight; Oligonucleotides/chemistry; Oligonucleotides/metabolism; Osmolar Concentration; RNA, Transfer/metabolism; Recombinant Proteins/chemistry; Recombinant Proteins/isolation & purification; Recombinant Proteins/metabolism; Substrate Specificity; Thermodynamics |
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Assay for RNase BN described in this paper, based on removal of the 3′-terminal mononucleotide AMP from the phage tRNA precursor analogue tRNA-C[14C]A. Synthesis of the substrate was described by Deutscher and Ghosh [1].
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