Mode of Action of RNase BN/RNase Z on tRNA Precursors

Abstract

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...

A comparison of key aspects of gene regulation in Streptomyces coelicolor and Escherichia coli using nucleotide‐resolution transcription maps produced in parallel by global and differential RNA sequencing

“…Studies of tRNA processing in E. coli have led to a model in which the mature 5′ end is generated by the ubiquitous endonuclease RNase P, and the mature 3′ end via endonucleolytic cleavage a few nucleotides downstream followed by 3′ exonucleolytic trimming to the CCA motif. The maturation of the 3′ end can be mediated by tRNase Z (RNase BN), which has dual endo/3′ to 5′ exonucleolytic activity (Dutta and Deutscher, 2010 ; Dutta et al ., 2012 ), or by the combined action of RNase E and 3′ to 5′ exonucleases, mainly RNases PH and T (Hartmann et al ., 2009 ). Consistent with this model, processing sites were identified at the precise 5′ end and within a few nucleotides downstream of the 3′ end of E. coli tRNAs (Fig.…”

The proteomic response to mutants of the Escherichia coli RNA degradosome

“…Since the coordination between RNA degradosome components organized by the C-terminal region of RNase E is required for decay of structured RNA substrates, it is tempting to assume that the observed increase in the cellular concentration of RNase R in cells expressing the truncated RNase E(1-602) (Figure 3) is due a defect in structured RNA observed in this strain. Similarly, RNase BN (Rbn) is implicated in the processing of tRNA precursors 27 . RNase BN abundance decreases in both the rhlB and pnp mutants (Figure 3), but is unchanged in rne(1-602) (or in the eno mutant).…”

Bacterial ribonucleases and their roles in RNA metabolism

“…RNase BN is also unusual in that its activity is the highest in the presence of Co 2+ (Asha et al 1983; Dutta and Deutscher 2009). In addition, Co 2+ promotes exoribonucleolytic activity against certain substrates, whereas Mg 2+ favors endoribonucleolytic activity (Dutta and Deutscher 2010). RNase BN is active on both double-and single-stranded RNA molecules, although the former are preferred (Dutta and Deutscher 2009).…”