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.
Highlights
TRNA genes in Escherichia coli are normally transcribed as precursor molecules that require processing at both their 5Јand 3Ј-ends to generate the mature functional forms [1]
Maturation of the 5Ј-end of tRNA precursors is carried out by a single endoribonuclease, RNase P, whereas 3Ј-processing generally is a multistep process requiring the action of both endoand exoribonucleases to remove the extra nucleotides following the encoded 3Ј-terminal -CCA sequence [1,2,3]
We discovered that rbn is nonessential in E. coli and that it encodes a polypeptide of 32.8 kDa [12]
Summary
Strains and Plasmids—Plasmid pBS(ϩ) (Stratagene) is an inducible multicopy vector. Plasmid pBSrbnϩ [12] was used to overexpress RNase BN. Unlabeled poly(A) used to dilute the radioactive material was obtained from Sigma. RNase BN Assay—RNase BN removes the 3Ј-terminal mononucleotide AMP from the phage tRNA precursor analogue tRNA-C[14C]A. Overexpression of RNase BN—Plasmid pBSrbnϩ carrying the rbn gene cloned under control of a T7 promoter was transformed into strain BL21(DE3) pLys. Bacteriophage DE3 contains the gene for T7 RNA polymerase controlled by the lacUV5 promoter, which is inducible by isopropyl--D-thiogalactopyranoside. Cells were grown to an absorbance of 0.5 at 600 nm in YT medium containing 200 g/ml ampicillin. To maintain the rbnϩ plasmid, cells were harvested by centrifugation at 5000 rpm for 5 min, washed with YT medium to remove -lactamase, and resuspended in fresh YT medium containing 300 g/ml ampicillin.
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