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Ciarán Condon

Publications and source records attributed to Ciarán Condon.

17 recordsLinked to original sources
PubMed2007

Maturation of the 5' end of Bacillus subtilis 16S rRNA by the essential ribonuclease YkqC/RNase J1.

Functional ribosomal RNAs are generated from longer precursor species in every organism known. Maturation of the 5' side of 16S rRNA in Escherichia coli is catalysed in a two-step process by the cooperative action of RNase E and RNase G. However, many bacteria lack RNase E and RNase G orthologues, raising the question as to how 16S rRNA processing occurs in these organisms. Here we show that the maturation of Bacillus subtilis 16S rRNA is also a two-step process and that the enzyme responsible for the generation of the mature 5' end is the widely distributed essential ribonuclease YkqC/RNase J1. Depletion of B. subtilis of RNase J1 results in an accumulation of 16S rRNA precursors in vivo. The precursor species are found in polysomes suggesting that they can function in translation. Mutation of the predicted catalytic site of RNase J1 abolishes both 16S rRNA processing and cell viability. Finally, purified RNase J1 can correctly mature precursor 16S rRNA assembled in 70S ribosomes, showing that its role is direct.

5' Flanking Region↗
PubMed2006

Shutdown decay of mRNA.

Although plasmid-borne and chromosomal toxin-antitoxin (TA) operons have been known for some time, the recent identification of mRNA as the target of at least two different classes of toxins has led to a dramatic renewal of interest in these systems as mediators of stress responses. Members of the MazF/PemK family, the so-called mRNA interferases, are ribonucleases that inhibit translation by destroying cellular mRNAs under stress conditions, while the founder member of the RelE family promotes cleavage of mRNAs through the ribosome. Detailed structures of these enzymes, often in complex with their inhibitors, have provided vital clues to their mechanisms of action. The primary role and regulation of these systems has been the subject of some controversy. One model suggests they play a beneficial role by wiping the slate clean and preventing wasteful energy consumption by the translational apparatus during adaptation to stress conditions, while another favours the idea that their main function is programmed cell death. The two models might not be mutually exclusive if a side-effect of prolonged exposure to toxic RNase activity without de novo synthesis of the inhibitor were a state of dormancy for which we do not yet understand the key to recovery. In this review, I discuss the recent developments in the rapidly expanding field of what I refer to as bacterial shutdown decay.

Adaptation, Biological↗
PubMed2006

Structure of the ubiquitous 3' processing enzyme RNase Z bound to transfer RNA.

The highly conserved ribonuclease RNase Z catalyzes the endonucleolytic removal of the 3' extension of the majority of tRNA precursors. Here we present the structure of the complex between Bacillus subtilis RNase Z and tRNA(Thr), the first structure of a ribonucleolytic processing enzyme bound to tRNA. Binding of tRNA to RNase Z causes conformational changes in both partners to promote reorganization of the catalytic site and tRNA cleavage.

Bacillus subtilis↗
PubMed2006

Type A and B RNase P RNAs are interchangeable in vivo despite substantial biophysical differences.

We show that structural type A and B bacterial ribonuclease P (RNase P) RNAs can fully replace each other in vivo despite the many reported differences in their biogenesis, biochemical/biophysical properties and enzyme function in vitro. Our findings suggest that many of the reported idiosyncrasies of type A and B enzymes either do not reflect the in vivo situation or are not crucial for RNase P function in vivo, at least under standard growth conditions. The discrimination of mature tRNA by RNase P, so far thought to prevent product inhibition of the enzyme in the presence of a large cellular excess of mature tRNA relative to the precursor form, is apparently not crucial for RNase P function in vivo.

Bacillus subtilis↗
PubMed2005

Sodium-induced GCN4 expression controls the accumulation of the 5' to 3' RNA degradation inhibitor, 3'-phosphoadenosine 5'-phosphate.

Most cytoplasmic mRNAs are decapped and digested by the 5'-3'-exonuclease Xrn1p in Saccharomyces cerevisiae. The activity of Xrn1p is naturally inhibited in the presence of 3'-phosphoadenosine 5'-phosphate (pAp), a metabolite produced during sulfate assimilation that is quickly metabolized to AMP by the enzymatic activity of Hal2p. However, pAp accumulates and 5'-3' degradation decreases in the presence of ions known to inhibit Hal2p activity, such as sodium or lithium. We have shown that yeast cells can better adapt to the presence of sodium than lithium because of their ability to reduce pAp accumulation by activating HAL2 expression in a Gcn4p-dependent response, a regulatory loop that is likely to be conserved in different yeast species. We have thus identified a new role for the transcriptional activity of Gcn4p in maintaining an active mRNA degradation pathway under conditions of sodium stress. Since deregulation of proteins involved in different metabolic pathways is observed in xrn1Delta mutants, the maintenance of mRNA degradation capacity is likely to be important for the accurate and rapid adaptation of gene expression to salt stress.

Adenosine Diphosphate↗
PubMed2005

The 5S rRNA maturase, ribonuclease M5, is a Toprim domain family member.

The maturation of 5S ribosomal RNA in low G+C Gram-positive bacteria is catalyzed by a highly conserved, approximately 190 residue, enzyme, called ribonuclease M5 (RNase M5). Sequence alignment had predicted that the N-terminal half of RNase M5 would consist of a Toprim domain, a protein fold found in type IA and type II topoisomerases, DnaG-like primases, OLD family nucleases and RecR proteins [L. Aravind, D. D. Leipe and E. V. Koonin (1998) Nucleic Acids Res., 26, 4205-4213]. Here, we present structural modelling data and a mutational analysis of RNase M5 that confirms this hypothesis. The N-terminal half of RNase M5 can be fitted to the Toprim domain of the DnaG catalytic core. Mutation of amino acid residues highly conserved among RNase M5 enzymes and members of the Toprim domain family showed that alteration of residues critical for topoisomerase and primase activity also had a dramatic effect on the cleavage of 5S rRNA precursor by RNase M5 both in vivo and in vitro. This suggests that the mechanisms of double-stranded RNA cleavage by RNase M5 and double-stranded DNA cleavage by members of the topoisomerase family are related.

Amino Acid Sequence↗
PubMed2005

Ribonuclease PH plays a major role in the exonucleolytic maturation of CCA-containing tRNA precursors in Bacillus subtilis.

In contrast to Escherichia coli, where all tRNAs have the CCA motif encoded by their genes, two classes of tRNA precursors exist in the Gram-positive bacterium Bacillus subtilis. Previous evidence had shown that ribonuclease Z (RNase Z) was responsible for the endonucleolytic maturation of the 3' end of those tRNAs lacking an encoded CCA motif, accounting for about one-third of its tRNAs. This suggested that a second pathway of tRNA maturation must exist for those precursors with an encoded CCA motif. In this paper, we examine the potential role of the four known exoribonucleases of B.subtilis, PNPase, RNase R, RNase PH and YhaM, in this alternative pathway. In the absence of RNase PH, precursors of CCA-containing tRNAs accumulate that are a few nucleotides longer than the mature tRNA species observed in wild-type strains or in the other single exonuclease mutants. Thus, RNase PH plays an important role in removing the last few nucleotides of the tRNA precursor in vivo. The presence of three or four exonuclease mutations in a single strain results in CCA-containing tRNA precursors of increasing size, suggesting that, as in E.coli, the exonucleolytic pathway consists of multiple redundant enzymes. Assays of purified RNase PH using in vitro-synthesized tRNA precursor substrates suggest that RNase PH is sensitive to the presence of a CCA motif. The division of labor between the endonucleolytic and exonucleolytic pathways observed in vivo can be explained by the inhibition of RNase Z by the CCA motif in CCA-containing tRNA precursors and by the inhibition of exonucleases by stable secondary structure in the 3' extensions of the majority of CCA-less tRNAs.

Bacillus subtilis↗
PubMed2005

Structural basis for substrate binding, cleavage and allostery in the tRNA maturase RNase Z.

Transfer RNAs (tRNAs) are synthesized as part of longer primary transcripts that require processing of both their 3' and 5' extremities in every living organism known. The 5' side is processed (matured) by the ubiquitously conserved endonucleolytic ribozyme, RNase P, whereas removal of the 3' tails can be either exonucleolytic or endonucleolytic. The endonucleolytic pathway is catalysed by an enzyme known as RNase Z, or 3' tRNase. RNase Z cleaves precursor tRNAs immediately after the discriminator base (the unpaired nucleotide 3' to the last base pair of the acceptor stem, used as an identity determinant by many aminoacyl-tRNA synthetases) in most cases, yielding a tRNA primed for addition of the CCA motif by nucleotidyl transferase. Here we report the crystal structure of Bacillus subtilis RNase Z at 2.1 A resolution, and propose a mechanism for tRNA recognition and cleavage. The structure explains the allosteric properties of the enzyme, and also sheds light on the mechanisms of inhibition by the CCA motif and long 5' extensions. Finally, it highlights the extraordinary adaptability of the metallo-hydrolase domain of the beta-lactamase family for the hydrolysis of covalent bonds.

Allosteric Regulation↗
PubMed2005

The Bacillus subtilis ydcDE operon encodes an endoribonuclease of the MazF/PemK family and its inhibitor.

Operons encoding stable toxins and their labile antidote are widespread in prokaryotes and play important roles in plasmid partitioning and cellular responses to stress. One such family of toxins MazF/ChpAK/PemK encodes an endoribonuclease that inactivates cellular mRNAs by cleaving them at specific, but frequently occurring sites. Here we show that the Bacillus subtilis ydcE gene encodes a member of this family of RNases, which we have called EndoA. Overexpression of EndoA is toxic for bacterial cell growth and this toxicity is reversed by coexpression of the gene immediately upstream, ydcD. Furthermore, YdcD inhibits EndoA activity directly in vitro. EndoA has similar cleavage specificity to MazF and PemK and yields cleavage products with 3'-phosphate and 5'-hydroxyl groups, typical of EDTA-resistant degradative RNases. This is the first example of an antitoxin-toxin system in B. subtilis.

Bacillus subtilis↗
PubMed2004

The continuing story of endoribonuclease III.

Endoribonuclease III (RNase III) plays an important role in the processing of rRNA and mRNAs. It is timely to summarize the most relevant insights obtained during the last years stemming from RNase III. With this aim, the present mini-review provides a wealth of new information focusing on the distribution and architecture of RNase III, substrate recognition, cleavage mechanisms and regulation of gene expression.

Bacillus subtilis↗
PubMed2003

Endonucleolytic processing of CCA-less tRNA precursors by RNase Z in Bacillus subtilis.

In contrast to Escherichia coli, where the 3' ends of tRNAs are primarily generated by exoribonucleases, maturation of the 3' end of tRNAs is catalysed by an endoribonuclease, known as RNase Z (or 3' tRNase), in many eukaryotic and archaeal systems. RNase Z cleaves tRNA precursors 3' to the discriminator base. Here we show that this activity, previously unsuspected in bacteria, is encoded by the yqjK gene of Bacillus subtilis. Decreased yqjK expression leads to an accumulation of a population of B.subtilis tRNAs in vivo, none of which have a CCA motif encoded in their genes, and YqjK cleaves tRNA precursors with the same specificity as plant RNase Z in vitro. We have thus renamed the gene rnz. A CCA motif downstream of the discriminator base inhibits RNase Z activity in vitro, with most of the inhibition due to the first C residue. Lastly, tRNAs with long 5' extensions are poor substrates for cleavage, suggesting that for some tRNAs, processing of the 5' end by RNase P may have to precede RNase Z cleavage.

Amino Acid Sequence↗
PubMed2003

RNA processing and degradation in Bacillus subtilis.

This review focuses on the enzymes and pathways of RNA processing and degradation in Bacillus subtilis, and compares them to those of its gram-negative counterpart, Escherichia coli. A comparison of the genomes from the two organisms reveals that B. subtilis has a very different selection of RNases available for RNA maturation. Of 17 characterized ribonuclease activities thus far identified in E. coli and B. subtilis, only 6 are shared, 3 exoribonucleases and 3 endoribonucleases. Some enzymes essential for cell viability in E. coli, such as RNase E and oligoribonuclease, do not have homologs in B. subtilis, and of those enzymes in common, some combinations are essential in one organism but not in the other. The degradation pathways and transcript half-lives have been examined to various degrees for a dozen or so B. subtilis mRNAs. The determinants of mRNA stability have been characterized for a number of these and point to a fundamentally different process in the initiation of mRNA decay. While RNase E binds to the 5' end and catalyzes the rate-limiting cleavage of the majority of E. coli RNAs by looping to internal sites, the equivalent nuclease in B. subtilis, although not yet identified, is predicted to scan or track from the 5' end. RNase E can also access cleavage sites directly, albeit less efficiently, while the enzyme responsible for initiating the decay of B. subtilis mRNAs appears incapable of direct entry. Thus, unlike E. coli, RNAs possessing stable secondary structures or sites for protein or ribosome binding near the 5' end can have very long half-lives even if the RNA is not protected by translation.

Bacillus subtilis↗
PubMed2002

The phylogenetic distribution of bacterial ribonucleases.

Ribonucleases play key, often essential, roles in cellular metabolism. Nineteen ribonuclease activities, from 22 different proteins, have so far been described in bacteria, the majority of them from either Escherichia coli or Bacillus subtilis. Here we examine the phylogenetic distribution of all of these ribonucleases in 50 eubacterial and archaeal species whose genomes have been completely sequenced, with particular emphasis on the endoribonucleases. Although some enzymes are very highly conserved throughout evolution, there appears to be no truly universal ribonuclease. While some organisms, like E.coli, have a large selection of ribonucleases, many with overlapping functions, others seem to have relatively few or have many that remain to be discovered.

Bacillus subtilis↗
PubMed2002

Transfer RNA-mediated antitermination in vitro.

The threonyl-tRNA synthetase gene (thrS) is a member of the T-box family of approximately 250 genes, found essentially in Gram-positive bacteria, regulated by a tRNA-dependent antitermination mechanism in response to starvation for the cognate amino acid. While interaction between uncharged tRNA and the untranslated leader region of these genes has been firmly established by genetic means, attempts to show this interaction or to reconstitute the antitermination mechanism in vitro using purified tRNAs have so far failed. In addition, a number of conserved sequences have been identified in the T-box leaders, for which no function has yet been assigned. This suggests that factors other than the tRNA are important for this type of control. Here we demonstrate tRNA-mediated antitermination for the first time in vitro, using the regulatory tRNA(Thr) isoacceptor isolated from Bacillus subtilis and a partially purified protein fraction. As predicted by the model, aminoacylation of tRNA(Thr(GGU)) with threonine completely abolishes its ability to act as an effector. The role of the partially purified protein fraction can be functionally substituted by high concentrations of spermidine. However, this polyamine does not play a significant role in the induction of thrS expression in vivo, suggesting that it is specific protein co-factors that promote T-box gene regulation in conjunction with uncharged tRNA.

Anticodon↗
PubMed2002

Ribonuclease M5 has few, if any, mRNA substrates in Bacillus subtilis.

In Bacillus subtilis, maturation of 5S rRNA is catalyzed by an enzyme called RNase M5. We searched for potential mRNA substrates for RNase M5 by gene array technology, based on the premise that most endonucleolytic cleavages have an effect on the stability of RNA and hence on steady-state levels of expression. Only a handful of genes had significantly altered expression in rnmV mutants compared to wild-type strains that could subsequently be confirmed by Northern blotting. The effect of RNase M5 on the expression of the best candidates, the odhAB and sucCD operons, is indirect, by a mechanism we do not yet understand. We show that an effect of RNase M5 on the expression of the remaining candidate, ctsR, is due to the failure to process the 5S rRNA contained in the rrnW lying directly upstream. We thus conclude that RNase M5 has very few or possibly no mRNA substrates in B. subtilis.

Bacillus subtilis↗