RNase BN, the Escherichia coli homolog of RNase Z, was previously shown to act as both a distributive exoribonuclease and an endoribonuclease on model RNA substrates and to be inhibited by the presence of a 3-terminal CCA sequence. Here, we examined the mode of action of RNase BN on bacteriophage and bacterial tRNA precursors, particularly in light of a recent report suggesting that RNase BN removes CCA sequences (Takaku, H., and Nashimoto, M. (2008) Genes Cells 13, 1087-1097). We show that purified RNase BN can process both CCAless and CCA-containing tRNA precursors. On CCA-less precursors, RNase BN cleaved endonucleolytically after the discriminator nucleotide to allow subsequent CCA addition. On CCA-containing precursors, RNase BN acted as either an exoribonuclease or endoribonuclease depending on the nature of the added divalent cation. Addition of Co 2؉ resulted in higher activity and predominantly exoribonucleolytic activity, whereas in the presence of Mg 2؉ , RNase BN was primarily an endoribonuclease. In no case was any evidence obtained for removal of the CCA sequence. Certain tRNA precursors were extremely poor substrates under any conditions tested. These findings provide important information on the ability of RNase BN to process tRNA precursors and help explain the known physiological properties of this enzyme. In addition, they call into question the removal of CCA sequences by RNase BN.3Ј-Maturation of tRNA precursors differs among organisms depending on whether or not the universal 3Ј-terminal CCA sequence is encoded (1-6). In eukaryotic cells, this sequence is absent from tRNA precursors, and the 3Ј-processing step is catalyzed by the endoribonuclease, RNase Z or 3Ј-tRNase, which cleaves at the discriminator base to allow subsequent CCA addition by tRNA nucleotidyltransferase (3, 5, 7). In contrast, in some bacteria, such as Escherichia coli, the CCA sequence is encoded, and maturation of the 3Ј terminus is carried out by exoribonucleases (8, 9), whereas in other organisms, such as Bacillus subtilis, both types of 3Ј-processing are operative (3). Surprisingly, in E. coli, despite the fact that the CCA sequence is present in all known tRNA precursors, an RNase Z homolog, termed RNase BN, is present (10). As a consequence, it has been of considerable interest to understand the function in E. coli of this apparently unnecessary enzyme.RNase BN was originally discovered as an enzyme required for the maturation of those bacteriophage T4 precursor tRNAs that lack a CCA sequence (11). Subsequently, RNase BN was shown to also be able to process the CCA-encoded host E. coli tRNA precursors in vivo (8, 9). However, of the five E. coli RNases able to carry out the 3Ј-processing reaction, RNase BN was the least efficient (12, 13). Thus, under normal growth conditions, RNase BN is unlikely to function in total tRNA maturation, although it cannot be ruled out that the enzyme may participate in 3Ј-maturation of some select tRNA species (8). However, because E. coli mutants lacking RNase BN grow essentially nor...