RNase BN Research Articles

A novel mechanism of RyeA/SraC induction under acid stress

RyeA/SraC is a cis-encoded small RNA (sRNA), which act as an anti-toxin to RpoS-regulated RyeB toxin in Escherichia coli. Ectopic expression of RyeA was reported to diminish the RyeB accumulation by serving as a RNA trap. Lower abundance of RyeA in the early exponential growth phase turned out to be the outcome of its degradation by RNase BN/Z. In the current study, we show that RyeA is an acid stress inducible sRNA, and global stress responsive factor RpoS appeared to be inessential in RyeA induction. Although, ryeB-pphA dicistronic transcript at low pH condition was stimulated by ∼4-fold, however, RyeB population was found to be decreased by > 50% under the same condition by the decoy action of enhanced RyeA accumulation. Investigation of the mechanism of RyeA induceduction at low pH in the exponential phase, revealed that RNase BN/Z, which catabolizes RyeA in the exponential phase, appeared to be highly sensitive to low pH stress. Both mRNA and protein level of RNase BN transpired to be decreased to <10% of their initial population. The expression of RyeA under acid stress is regulated by a feed-forward mechanism to normalize the RyeB profusion.

A novel mechanism of ribonuclease regulation: GcvB and Hfq stabilize the mRNA that encodes RNase BN/Z during exponential phase

RNase BN, the Escherichia coli RNase Z family member, plays a limited role in tRNA metabolism, in contrast to most other organisms. However, RNase BN does act on 6S RNA, the global transcription regulator, degrading it in exponential-phase cells and maintaining it at low levels during this phase of growth. RNase BN levels decrease in stationary-phase cells, leading to elevation of 6S RNA and subsequent regulation of RNA polymerase. These findings were the first indication that RNase BN itself is growth phase-regulated. Here, we analyze the mechanism of this regulation of RNase BN. We find that RNase BN decreases in stationary phase because its mRNA becomes unstable, due primarily to its degradation by RNase E. However, in exponential-phase cells rbn mRNA is stabilized due to binding by the sRNA, GcvB, and the protein, Hfq, which reduce cleavage by RNase E. Because the amount of GcvB decreases in stationary phase, rbn mRNA is less protected and becomes increasingly unstable resulting in reduction in the amount of RNase BN. The small RNA-dependent, positive regulation of RNase BN in exponential-phase cells is the first example of this novel mechanism for RNase regulation.

Regulation of RyeA/SraC expression in Escherichia coli

Small RNAs (sRNAs) play a central role in regulating almost all physiological processes in bacteria. Majority of those sRNAs base pair with their targets and modulate their expressions. RyeA, previously known as SraC in Escherichia coli, is transcribed from a DNA strand complementary to the one from which another stationary phase induced sRNA RyeB/SdsR is synthesised. RyeA and RyeB in the stationary phase constitute a toxin-antitoxin system where RyeA normalizes accumulation of RyeB toxin by acting as RNA sponge. Aside from that, no more information is known about the regulation of RyeA expression. In the current study, we have systematically investigated the regulation of RyeA expression in different growth phases, and identified that RyeA expression is regulated neither by stationary phase-specific σ-factor nor by RNA chaperon Hfq. A dual function ribonuclease RNase BN mitigate its expression in the exponential phase. Thus, deletion of rbn gene promoted the stability of RyeA in the exponential phase. Conversely, RyeB in the stationary phase act as RNA decoy leading to RyeA degradation, and consequently, the preclusion of RyeB in the E. coli genome elevated RyeA. These regulatory mechanisms will help identify the primary role of RyeA in E. coli.

Growth Phase-dependent Variation of RNase BN/Z Affects Small RNAs: REGULATION OF 6S RNA

RNase BN, the RNase Z family member in E. coli, can participate in the processing of tRNA precursors. However, this function only becomes apparent when other processing enzymes are absent, raising the question of its primary physiological role. Here, we show that RNase BN itself is subject to growth phase-dependent regulation, because both rbn mRNA and RNase BN protein are at their highest levels in early exponential phase, but then decrease dramatically and are essentially absent in stationary phase. As a consequence of this variation, certain small RNAs, such as 6S RNA, remain low in exponential phase cells, and increase greatly in stationary phase. RNase BN affects 6S RNA abundance by decreasing its stability in exponential phase. RNase BN levels increase rapidly as cells exit stationary phase and are primarily responsible for the decrease in 6S RNA that accompanies this process. Purified RNase BN directly cleaves 6S RNA as shown by in vitro assays, and the 6S RNA:pRNA duplex is an even more favorable substrate of RNase BN. The exoribonuclease activity of RNase BN is unnecessary because all its action on 6S RNA is due to endonucleolytic cleavages. These data indicate that RNase BN plays an important role in determining levels of the global transcription regulator, 6S RNA, throughout the growth cycle.

Endonucleolytic cleavages by RNase E generate the mature 3' termini of the three proline tRNAs in Escherichia coli.

We demonstrate here for the first time that proline tRNA 3′ end maturation in Escherichia coli employs a one-step endonucleolytic pathway that does not involve any of the six 3′ → 5′ exonucleases (RNase T, RNase PH, RNase D, RNase BN, RNase II and polynucleotide phosphorylase [PNPase]) to generate the mature CCA terminus. Rather, RNase E is primarily responsible for the endonucleolytic removal of the entire Rho-independent transcription terminator associated with the proK, proL and proM primary transcripts by cleaving immediately downstream of the CCA determinant. In the absence of RNase E, RNase G and RNase Z are weakly able to process the proK and proM transcripts, while PNPase and RNase P are utilized in the processing of proL. The terminator fragment derived from the endonucleolytic cleavage of proL transcript is degraded through a PNPase-dependent pathway. It is not clear which enzymes degrade the proK and proM terminator fragments. Our data also suggest that the mature 5′ nucleotide of the proline tRNAs may be responsible for the cleavage specificity of RNase E at the 3′ terminus.

Exoribonuclease and Endoribonuclease Activities of RNase BN/RNase Z both Function in Vivo

Escherichia coli RNase BN, a member of the RNase Z family of endoribonucleases, differs from other family members in that it also can act as an exoribonuclease in vitro. Here, we examine whether this activity of RNase BN also functions in vivo. Comparison of the x-ray structure of RNase BN with that of Bacillus subtilis RNase Z, which lacks exoribonuclease activity, revealed that RNase BN has a narrower and more rigid channel downstream of the catalytic site. We hypothesized that this difference in the putative RNA exit channel might be responsible for the acquisition of exoribonuclease activity by RNase BN. Accordingly, we generated several mutant RNase BN proteins in which residues within a loop in this channel were converted to the corresponding residues present in B. subtilis RNase Z, thus widening the channel and increasing its flexibility. The resulting mutant RNase BN proteins had reduced or were essentially devoid of exoribonuclease activity in vitro. Substitution of one mutant rbn gene (P142G) for wild type rbn in the E. coli chromosome revealed that the exoribonuclease activity of RNase BN is not required for maturation of phage T4 tRNA precursors, a known specific function of this RNase. On the other hand, removal of the exoribonuclease activity of RNase BN in a cell lacking other processing RNases leads to slower growth and affects maturation of multiple tRNA precursors. These findings help explain how RNase BN can act as both an exo- and an endoribonuclease and also demonstrate that its exoribonuclease activity is capable of functioning in vivo, thus widening the potential role of this enzyme in E. coli.

Mode of Action of RNase BN/RNase Z on tRNA Precursors: RNase BN DOES NOT REMOVE THE CCA SEQUENCE FROM tRNA

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 CCA-less 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.

Catalytic Properties of RNase BN/RNase Z from Escherichia coli

Processing of the 3' terminus of tRNA in many organisms is carried out by an endoribonuclease termed RNase Z or 3'-tRNase, which cleaves after the discriminator nucleotide to allow addition of the universal -CCA sequence. In some eubacteria, such as Escherichia coli, the -CCA sequence is encoded in all known tRNA genes. Nevertheless, an RNase Z homologue (RNase BN) is still present, even though its action is not needed for tRNA maturation. To help identify which RNA molecules might be potential substrates for RNase BN, we carried out a detailed examination of its specificity and catalytic potential using a variety of synthetic substrates. We show here that RNase BN is active on both double- and single-stranded RNA but that duplex RNA is preferred. The enzyme displays a profound base specificity, showing no activity on runs of C residues. RNase BN is strongly inhibited by the presence of a 3'-CCA sequence or a 3'-phosphoryl group. Digestion by RNase BN leads to 3-mers as the limit products, but the rate slows on molecules shorter than 10 nucleotides in length. Most interestingly, RNase BN acts as a distributive exoribonuclease on some substrates, releasing mononucleotides and a ladder of digestion products. However, RNase BN also cleaves endonucleolytically, releasing 3' fragments as short as 4 nucleotides. Although the presence of a 3'-phosphoryl group abolishes exoribonuclease action, it has no effect on the endoribonucleolytic cleavages. These data suggest that RNase BN may differ from other members of the RNase Z family, and they provide important information to be considered in identifying a physiological role for this enzyme.

The RNase Z Homologue Encoded by Escherichia coli elaC Gene Is RNase BN

In eukaryotes, archaea, and in some eubacteria, removal of 3' precursor sequences during maturation of tRNA is catalyzed by an endoribonuclease, termed RNase Z. In contrast, in Escherichia coli, a variety of exoribonucleases carry out final 3' maturation. Yet, E. coli retains an RNase Z homologue, ElaC, whose function is under active study. We have overexpressed and purified to homogeneity His-tagged ElaC and show here that it is, in fact, the previously described enzyme, RNase BN. Thus, purified ElaC displays structural and catalytic properties identical to those ascribed to RNase BN. In addition, an elaC mutant strain behaves identically to a known RNase BN- strain, CAN. Finally, we show that wild type elaC can complement the mutation in strain CAN and that the elaC gene in strain CAN carries a nonsense mutation that results in loss of RNase BN activity. These data correct a previous misassignment for the gene encoding RNase BN. Based on the fact that the original RNase BN mutation has now been identified, we propose that the elaC gene be renamed rbn.

Exoribonucleases and Endoribonucleases.

This review provides a description of the known Escherichia coli ribonucleases (RNases), focusing on their structures, catalytic properties, genes, physiological roles, and possible regulation. Currently, eight E. coli exoribonucleases are known. These are RNases II, R, D, T, PH, BN, polynucleotide phosphorylase (PNPase), and oligoribonuclease (ORNase). Based on sequence analysis and catalytic properties, the eight exoribonucleases have been grouped into four families. These are the RNR family, including RNase II and RNase R; the DEDD family, including RNase D, RNase T, and ORNase; the RBN family, consisting of RNase BN; and the PDX family, including PNPase and RNase PH. Seven well-characterized endoribonucleases are known in E. coli. These are RNases I, III, P, E, G, HI, and HII. Homologues to most of these enzymes are also present in Salmonella. Most of the endoribonucleases cleave RNA in the presence of divalent cations, producing fragments with 3'-hydroxyl and 5'-phosphate termini. RNase H selectively hydrolyzes the RNA strand of RNA?DNA hybrids. Members of the RNase H family are widely distributed among prokaryotic and eukaryotic organisms in three distinct lineages, RNases HI, HII, and HIII. It is likely that E. coli contains additional endoribonucleases that have not yet been characterized. First of all, endonucleolytic activities are needed for certain known processes that cannot be attributed to any of the known enzymes. Second, homologues of known endoribonucleases are present in E. coli. Third, endonucleolytic activities have been observed in cell extracts that have different properties from known enzymes.

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 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.

Identification and characterization of the Escherichia coli rbn gene encoding the tRNA processing enzyme RNase BN.

The gene encoding RNase BN was localized to 88 min on the Escherichia coli chromosome by a novel suppressor assay and conjugational and transductional analysis. Assay of subclones derived from lambda phage 543 of the Kohara library, which encompasses this region of the chromosome, for elevated RNase BN activity identified o290, a previously reported open reading frame, as the gene encoding RNase BN. Interruption of this gene with a Kan(r) cassette and introduction into the chromosome eliminated cellular RNase BN activity but had no effect on cell growth. On the basis of these data, we suggest that o290 be renamed rbn. Potential homologs of rbn in other organisms also were identified.

Multiple exoribonucleases are required for the 3' processing of Escherichia coli tRNA precursors in vivo.

Our knowledge of the 3' processing of tRNA precursors is severely limited. Although six exoribonucleases able to act on Escherichia coli tRNA precursors in vitro have been identified, their involvement in tRNA maturation in vivo has not been demonstrated. Here we show, using a wide range of multiple RNase-deficient strains and a quantitative suppression assay, that at least five of these enzymes--RNase II, RNase D, RNase BN, RNase T, and RNase PH--can participate in the synthesis of functional tRNA(Tyr)su+3 in vivo. Moreover, any one of the five RNases is sufficient to allow tRNA processing to proceed although with varying effectiveness. Examination of the level of aminoacylation of tRNA isolated from RNase-deficient strains suggested that tRNA precursors accumulate in the most defective cells. These data indicate that exoribonucleases are required for tRNA maturation in vivo and that there is a high degree of functional overlap among the enzymes. These studies contribute to the identification of all the enzymes necessary for defining the complete processing pathway for E. coli tRNA precursors.

The presence of only one of five exoribonucleases is sufficient to support the growth of Escherichia coli.

Escherichia coli contains multiple exoribonucleases. Strains lacking the exoribonucleases RNase II, D, BN, T, and PH are inviable. The introduction of a chromosomal, wild-type copy of the gene for any one of these enzymes is sufficient to allow cell growth, with the enzymes being in the following order of effectiveness: RNase T > RNase PH > RNase D > RNase II > RNase BN. The data indicate that these five exoribonucleases functionally overlap in vivo and that any one of them can take over the functions of all the others, although with various efficiencies.

A multiple mutant of Escherichia coli lacking the exoribonucleases RNase II, RNase D, and RNase BN.

A multiple mutant strain of Escherichia coli containing mutations affecting the exoribonucleases, RNase II, RNase D, and RNase BN, and also the endonuclease, RNase I, was constructed by P1-mediated transduction. Extracts of the mutant strain were lacking the aforementioned RNase activities. The multiple mutant displayed normal growth in both rich and minimal media at a variety of temperatures, recovered from starvation essentially as the wild-type parent, and could support the growth of a variety of bacteriophages. In addition, RNA synthesis was normal and no precursor RNA accumulation was observed. The properties of the mutant strain indicate that the three exoribonucleases are not essential for the viability of E. coli. The implications of these findings to our understanding of RNA processing and degradation are discussed.

Ribonuclease T: new exoribonuclease possibly involved in end-turnover of tRNA.

Examination of double mutants lacking one of the exoribonucleases, RNase II, RNase D, RNase BN, or RNase R, and also devoid of tRNA nucleotidyltransferase has suggested that none of these RNases participates in the end-turnover of tRNA. This prompted a search for and identification of a new exoribonuclease, termed RNase T. RNase T could be detected in mutant Escherichia coli strains lacking as many as three of the known exoribonucleases, and it could be separated from each of the four previously described RNases. RNase T is optimally active at pH 8-9 and requires a divalent cation for activity. The enzyme is sensitive to ionic strengths greater than 50 mM and is rapidly inactivated by heating at 45 degrees C. Its preferred substrate is tRNA-C-C-[14C]A, with much less activity shown against tRNA-C-C. RNase T is an exoribonuclease that initiates attack at the 3' hydroxyl terminus of tRNA and releases AMP in a random mode of hydrolysis. The possible involvement of RNase T in end-turnover of tRNA and in RNA metabolism in general are discussed.

Ribonuclease BN: identification and partial characterization of a new tRNA processing enzyme.

A new ribonuclease, RNase BN, has been identified and partially purified from a strain of Escherichia coli lacking RNase II and 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.