Purification and characterization of the tRNA-processing enzyme RNase BN

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 similar to 65 kDa. RNase BN displays a number of unusual catalytic prop erties compared with the other exoribonucleases of E. coli, The enzyme is m ost active at PH 6.5 in the presence of Co2+ and high concentrations of mon ovalent salts. It is highly specific for tRNA substrates containing an inco rrect 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 essent ially inactive as substrates, RNA or DNA oligonucleotides also are not subs trates, 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 B N in uninfected cells remains to be determined.