A new ribonuclease, RNase BN, has been identified and partially purified from a strain of Escherichia coli lacking RNase Hand RNase D by using the artificial tRNA precursor tRNA-C-[14C]U as substrate. This enzyme is present in E. coli B but absent from the tRNA processing mutant strain BN which is unable to process extraneous 3' residues on certain phage T4-specified tRNA precursors. The properties of RNase BN clearly distinguish this enzyme from other known E. coli exoribonucleases. It is optimally active at pH 6.5 with 0.2 mM divalent cation and 0.2 M monovalent cation. It is most active against tRNA substrates containing nucleotide substitutions within the -C-C-A sequence and relatively inactive against other types of RNAs. This substrate specificity in vitro is consistent with a processing function in vivo. However, in contrast to the other processing enzymes whose function has been confirmed by mutation, RNase BN is an exoribonuclease. The presence of multiple RNases in E. coli and a strategy for their identification and separation are discussed.Although considerable progress has been made in elucidating the overall pathway of tRNA processing, as yet there is relatively little information available about the nucleases involved, particularly with regard to processing at the 3' terminus (1). Earlier reports from our laboratory have described the identification, purification, and characterization of one exonuclease, RNase D, which has the in vitro properties expected for the processing nuclease that would remove extra residues following the -C-C-A sequence in Escherichia coli tRNA precursors (2-6). However, more recent experiments with E. coli strains deficient in RNase D have raised uncertainties about its actual physiological role (7,8).The existence of a second tRNA processing exoribonuclease has been suggested by the isolation of an E. coli mutant strain, termed BN (9). This strain is affected in its ability to process the 3' terminus of certain tRNA precursors synthesized after bacteriophage T4 infection (10), although growth of the host is not impaired (unpublished data). The affected T4 precursors lack all or part of the usual -C-C-A sequence and require removal of extraneous nucleotides prior to -C-C-A completion. Schmidt and McClain suggested that this processing defect was due to the loss of a specific enzyme, termed "BN ribonuclease" (11). Although the enzyme described by these workers was not characterized in detail, the properties that were reported were identical to those now attributed to RNase D (5), rather than to a new enzyme, raising questions about the existence of a distinct BN ribonuclease. However, further genetic and biochemical studies in our laboratory have shown that the mutation in strain BN is distinct from RNase D (8, 12), encouraging us to search further for the enzyme affected by the BN mutation.Establishing the existence of RNase BN has been hampered by the large amounts of RNase II and RNase D generally found in extracts, which tend to mask other less-active enzymes u...

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Flumazenil effectively reverses the benzodiazepine component of ventilatory depression during combined administration of a benzodiazepine and an opioid.

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