RESUMO
The pathways for ribosomal RNA (rRNA) maturation diverge greatly among the domains of life. In the Gram-positive model bacterium, Bacillus subtilis, the final maturation steps of the two large ribosomal subunit (50S) rRNAs, 23S and 5S pre-rRNAs, are catalyzed by the double-strand specific ribonucleases (RNases) Mini-RNase III and RNase M5, respectively. Here we present a protocol that allowed us to solve the 3.0 and 3.1 Å resolution cryoelectron microscopy structures of these RNases poised to cleave their pre-rRNA substrates within the B. subtilis 50S particle. These data provide the first structural insights into rRNA maturation in bacteria by revealing how these RNases recognize and process double-stranded pre-rRNA. Our structures further uncover how specific ribosomal proteins act as chaperones to correctly fold the pre-rRNA substrates and, for Mini-III, anchor the RNase to the ribosome. These r-proteins thereby serve a quality-control function in the process from accurate ribosome assembly to rRNA processing.
Assuntos
Bacillus subtilis/enzimologia , Proteínas de Bactérias/química , Precursores de RNA/metabolismo , Ribonucleases/química , Subunidades Ribossômicas Maiores de Bactérias/metabolismo , Bacillus subtilis/ultraestrutura , Proteínas de Bactérias/ultraestrutura , Sequência de Bases , Microscopia Crioeletrônica , Modelos Moleculares , Precursores de RNA/ultraestrutura , Ribonucleases/ultraestrutura , Subunidades Ribossômicas Maiores de Bactérias/ultraestrutura , Especificidade por SubstratoRESUMO
rRNAs and tRNAs universally require processing from longer primary transcripts to become functional for translation. Here, we describe an unsuspected link between tRNA maturation and the 3' processing of 16S rRNA, a key step in preparing the small ribosomal subunit for interaction with the Shine-Dalgarno sequence in prokaryotic translation initiation. We show that an accumulation of either 5' or 3' immature tRNAs triggers RelA-dependent production of the stringent response alarmone (p)ppGpp in the Gram-positive model organism Bacillus subtilis. The accumulation of (p)ppGpp and accompanying decrease in GTP levels specifically inhibit 16S rRNA 3' maturation. We suggest that cells can exploit this mechanism to sense potential slowdowns in tRNA maturation and adjust rRNA processing accordingly to maintain the appropriate functional balance between these two major components of the translation apparatus.
Assuntos
Bacillus subtilis/genética , Regulação Bacteriana da Expressão Gênica , Guanosina Pentafosfato/biossíntese , Iniciação Traducional da Cadeia Peptídica , RNA Ribossômico 16S/genética , RNA de Transferência/genética , Bacillus subtilis/metabolismo , Sequência de Bases , Guanosina Pentafosfato/genética , Guanosina Trifosfato/metabolismo , Ligases/genética , Ligases/metabolismo , Conformação de Ácido Nucleico , RNA Ribossômico 16S/química , RNA Ribossômico 16S/metabolismo , RNA de Transferência/química , RNA de Transferência/metabolismo , Subunidades Ribossômicas Maiores de Bactérias/genética , Subunidades Ribossômicas Maiores de Bactérias/metabolismo , Subunidades Ribossômicas Menores de Bactérias/genética , Subunidades Ribossômicas Menores de Bactérias/metabolismoRESUMO
Cellular processes require precise and specific gene regulation, in which continuous mRNA degradation is a major element. The mRNA degradation mechanisms should be able to degrade a wide range of different RNA substrates with high efficiency, but should at the same time be limited, to avoid killing the cell by elimination of all cellular RNA. RNase Y is a major endoribonuclease found in most Firmicutes, including Bacillus subtilis and Staphylococcus aureus. However, the molecular interactions that direct RNase Y to cleave the correct RNA molecules at the correct position remain unknown. In this work we have identified transcripts that are homologs in S. aureus and B. subtilis, and are RNase Y targets in both bacteria. Two such transcript pairs were used as models to show a functional overlap between the S. aureus and the B. subtilis RNase Y, which highlighted the importance of the nucleotide sequence of the RNA molecule itself in the RNase Y targeting process. Cleavage efficiency is driven by the primary nucleotide sequence immediately downstream of the cleavage site and base-pairing in a secondary structure a few nucleotides downstream. Cleavage positioning is roughly localised by the downstream secondary structure and fine-tuned by the nucleotide immediately upstream of the cleavage. The identified elements were sufficient for RNase Y-dependent cleavage, since the sequence elements from one of the model transcripts were able to convert an exogenous non-target transcript into a target for RNase Y.
Assuntos
Bacillus subtilis , Regulação Bacteriana da Expressão Gênica , Clivagem do RNA , Estabilidade de RNA , RNA Bacteriano , Staphylococcus aureus , Staphylococcus aureus/genética , Staphylococcus aureus/enzimologia , Bacillus subtilis/genética , Bacillus subtilis/enzimologia , Bacillus subtilis/metabolismo , RNA Bacteriano/metabolismo , RNA Bacteriano/genética , Estabilidade de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Endorribonucleases/metabolismo , Endorribonucleases/genética , Conformação de Ácido Nucleico , Sequência de BasesRESUMO
Rae1 is a well-conserved endoribonuclease among Gram-positive bacteria, cyanobacteria, and the chloroplasts of higher plants. We have previously shown that Rae1 cleaves the Bacillus subtilis yrzI operon mRNA in a translation-dependent manner within a short open reading frame (ORF) called S1025, encoding a 17-amino acid (aa) peptide of unknown function. Here, we map a new Rae1 cleavage site in the bmrBCD operon mRNA encoding a multidrug transporter, within an unannotated 26-aa cryptic ORF that we have named bmrX Expression of the bmrCD portion of the mRNA is ensured by an antibiotic-dependent ribosome attenuation mechanism within the upstream ORF bmrB Cleavage by Rae1 within bmrX suppresses bmrCD expression that escapes attenuation control in the absence of antibiotics. Similar to S1025, Rae1 cleavage within bmrX is both translation- and reading frame-dependent. Consistent with this, we show that translation-dependent cleavage by Rae1 promotes ribosome rescue by the tmRNA.
Assuntos
Endorribonucleases , Biossíntese de Proteínas , Endorribonucleases/genética , Endorribonucleases/metabolismo , Fases de Leitura Aberta , Ribossomos/genética , Ribossomos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismoRESUMO
RNase J1 is the major 5'-to-3' bacterial exoribonuclease. We demonstrate that in its absence, RNA polymerases (RNAPs) are redistributed on DNA, with increased RNAP occupancy on some genes without a parallel increase in transcriptional output. This suggests that some of these RNAPs represent stalled, non-transcribing complexes. We show that RNase J1 is able to resolve these stalled RNAP complexes by a "torpedo" mechanism, whereby RNase J1 degrades the nascent RNA and causes the transcription complex to disassemble upon collision with RNAP. A heterologous enzyme, yeast Xrn1 (5'-to-3' exonuclease), is less efficient than RNase J1 in resolving stalled Bacillus subtilis RNAP, suggesting that the effect is RNase-specific. Our results thus reveal a novel general principle, whereby an RNase can participate in genome-wide surveillance of stalled RNAP complexes, preventing potentially deleterious transcription-replication collisions.
Assuntos
Bacillus subtilis/enzimologia , Exorribonucleases/metabolismo , RNA Mensageiro/metabolismo , Bacillus subtilis/genética , Proteínas de Bactérias/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Regulação Bacteriana da Expressão Gênica , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA Mensageiro/genética , Transcrição GênicaRESUMO
Quick growth restart after upon encountering favourable environmental conditions is a major fitness contributor in natural environment. It is widely assumed that the time required to restart growth after nutritional upshift is determined by how long it takes for cells to synthesize enough ribosomes to produce the proteins required to reinitiate growth. Here we show that a reduction in the capacity to synthesize ribosomes by reducing number of ribosomal RNA (rRNA) operons (rrn) causes a longer transition from stationary phase to growth of Escherichia coli primarily due to high mortality rates. Cell death results from DNA replication blockage and massive DNA breakage at the sites of the remaining rrn operons that become overloaded with RNA polymerases (RNAPs). Mortality rates and growth restart duration can be reduced by preventing R-loop formation and improving DNA repair capacity. The same molecular mechanisms determine the duration of the recovery phase after ribosome-damaging stresses, such as antibiotics, exposure to bile salts or high temperature. Our study therefore suggests that a major function of rrn operon multiplicity is to ensure that individual rrn operons are not saturated by RNAPs, which can result in catastrophic chromosome replication failure and cell death during adaptation to environmental fluctuations.
The ability to modulate translation capacity, which resides greatly on a number of ribosomes, provides robustness in fluctuating environments. Because translation is energetically the most expensive process in cells, cells must constantly adapt the rate of ribosome production to resource availability. This is primarily achieved by regulating ribosomal RNA (rRNA) synthesis, to which ribosomal proteins synthesis is adjusted. The multiplicity of rRNA encoding operons per bacterial genome exceeds requirements for the maximal growth rates in non-stress conditions. In this study, the authors provide evidence that a major function of rRNA operon multiplicity is to ensure that individual operons are not saturated by RNA polymerases during adaptation to environmental fluctuations, which can result in catastrophic chromosome replication failure and cell death.
Assuntos
Genoma Bacteriano , Óperon de RNAr , Escherichia coli/metabolismo , Óperon , Ribossomos/genética , Ribossomos/metabolismo , RNA Bacteriano/genética , RNA Ribossômico/genética , RNA Ribossômico/metabolismo , Instabilidade GenômicaRESUMO
sRNAs are a taxonomically-restricted but transcriptomically-abundant class of post-transcriptional regulators. While of major importance for adaption to the environment, we currently lack global-scale methodology enabling target identification, especially in species without known RNA hub proteins (e.g. Hfq). Using psoralen RNA cross-linking and Illumina-sequencing we identify RNA-RNA interacting pairs in vivo in Bacillus subtilis, resolving previously well-described interactants. Although sRNA-sRNA pairings are rare (compared with sRNA-mRNA), we identify a robust example involving the conserved sRNA RoxS and an unstudied sRNA RosA (Regulator of sRNA A). We show RosA to be the first confirmed RNA sponge described in a Gram-positive bacterium. RosA interacts with at least two sRNAs, RoxS and FsrA. The RosA/RoxS interaction not only affects the levels of RoxS but also its processing and regulatory activity. We also found that the transcription of RosA is repressed by CcpA, the key regulator of carbon-metabolism in B. subtilis. Since RoxS is already known to be transcriptionally controlled by malate via the transcriptional repressor Rex, its post-transcriptional regulation by CcpA via RosA places RoxS in a key position to control central metabolism in response to varying carbon sources.
Assuntos
Bacillus subtilis/genética , RNA Bacteriano/metabolismo , Pequeno RNA não Traduzido/metabolismo , Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Carbono/metabolismo , Aptidão Genética , Proteoma , Processamento Pós-Transcricional do RNA , Estabilidade de RNA , Pequeno RNA não Traduzido/biossíntese , Pequeno RNA não Traduzido/genética , Pequeno RNA não Traduzido/fisiologia , Transcrição GênicaRESUMO
The PIN domain plays a central role in cellular RNA biology and is involved in processes as diverse as rRNA maturation, mRNA decay and telomerase function. Here, we solve the crystal structure of the Rae1 (YacP) protein of Bacillus subtilis, a founding member of the NYN (Nedd4-BP1/YacP nuclease) subfamily of PIN domain proteins, and identify potential substrates in vivo Unexpectedly, degradation of a characterised target mRNA was completely dependent on both its translation and reading frame. We provide evidence that Rae1 associates with the B. subtilis ribosome and cleaves between specific codons of this mRNA in vivo Critically, we also demonstrate translation-dependent Rae1 cleavage of this substrate in a purified translation assay in vitro Multiple lines of evidence converge to suggest that Rae1 is an A-site endoribonuclease. We present a docking model of Rae1 bound to the B. subtilis ribosomal A-site that is consistent with this hypothesis and show that Rae1 cleaves optimally immediately upstream of a lysine codon (AAA or AAG) in vivo.
Assuntos
Bacillus subtilis/enzimologia , Bacillus subtilis/metabolismo , Endorribonucleases/química , Endorribonucleases/metabolismo , Biossíntese de Proteínas , Estabilidade de RNA , Ribossomos/metabolismo , Cristalografia por Raios X , Modelos Biológicos , Modelos Moleculares , Conformação ProteicaRESUMO
All species transcribe ribosomal RNA in an immature form that requires several enzymes for processing into mature rRNA. The number and types of enzymes utilized for these processes vary greatly between different species. In low G + C Gram-positive bacteria including Bacillus subtilis and Geobacillus stearothermophilus, the endoribonuclease (RNase) M5 performs the final step in 5S rRNA maturation, by removing the 3'- and 5'-extensions from precursor (pre) 5S rRNA. This cleavage activity requires initial complex formation between the pre-rRNA and a ribosomal protein, uL18, making the full M5 substrate a ribonucleoprotein particle (RNP). M5 contains a catalytic N-terminal Toprim domain and an RNA-binding C-terminal domain, respectively, shown to assist in processing and binding of the RNP. Here, we present structural data that show how two Mg2+ ions are accommodated in the active site pocket of the catalytic Toprim domain and investigate the importance of these ions for catalysis. We further perform solution studies that support the previously proposed 3'-before-5' order of removal of the pre-5S rRNA extensions and map the corresponding M5 structural rearrangements during catalysis.
Assuntos
Bacillus subtilis/enzimologia , Endorribonucleases/química , Endorribonucleases/metabolismo , Geobacillus stearothermophilus/enzimologia , Magnésio/metabolismo , Precursores de RNA/metabolismo , RNA de Cadeia Dupla/metabolismo , RNA Ribossômico 5S/metabolismo , Sequência de Aminoácidos , Endorribonucleases/genética , Conformação de Ácido Nucleico , Precursores de RNA/genética , RNA de Cadeia Dupla/genética , RNA Ribossômico 5S/genética , Ribossomos/genética , Ribossomos/metabolismo , Especificidade por SubstratoRESUMO
Ribosomal RNAs are processed from primary transcripts containing 16S, 23S and 5S rRNAs in most bacteria. Maturation generally occurs in a two-step process, consisting of a first crude separation of the major species by RNase III during transcription, followed by precise trimming of 5' and 3' extensions on each species upon accurate completion of subunit assembly. The various endo- and exoribonucleases involved in the final processing reactions are strikingly different in Escherichia coli and Bacillus subtilis, the two best studied representatives of Gram-negative and Gram-positive bacteria, respectively. Here, we show that the one exception to this rule is the protein involved in the maturation of the 3' end of 16S rRNA. Cells depleted for the essential B. subtilis YqfG protein, a homologue of E. coli YbeY, specifically accumulate 16S rRNA precursors bearing 3' extensions. Remarkably, the essential nature of YqfG can be suppressed by deleting the ribosomal RNA degrading enzyme RNase R, i.e. a ΔyqfG Δrnr mutant is viable. Our data suggest that 70S ribosomes containing 30S subunits with 3' extensions of 16S rRNA are functional to a degree, but become substrates for degradation by RNase R and are eliminated.
Assuntos
Bacillus subtilis/genética , Proteínas de Bactérias/genética , Exorribonucleases/genética , Deleção de Genes , Processamento de Terminações 3' de RNA , RNA Ribossômico 16S/genética , Sequência de Aminoácidos , Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Sequência de Bases , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Exorribonucleases/metabolismo , Metaloproteínas/genética , Metaloproteínas/metabolismo , Precursores de RNA/genética , Precursores de RNA/metabolismo , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA Ribossômico 16S/metabolismo , Homologia de Sequência de Aminoácidos , Especificidade da EspécieRESUMO
In Escherichia coli, RNA degradation often begins with conversion of the 5'-terminal triphosphate to a monophosphate, creating a better substrate for internal cleavage by RNase E. Remarkably, no homolog of this key endonuclease is present in many bacterial species, such as Bacillus subtilis and various pathogens. Here, we report that the degradation of primary transcripts in B. subtilis can nevertheless be triggered by an analogous process to generate a short-lived, monophosphorylated intermediate. Like its E. coli counterpart, the B. subtilis RNA pyrophosphohydrolase that catalyzes this event is a Nudix protein that prefers unpaired 5' ends. However, in B. subtilis, this modification exposes transcripts to rapid 5' exonucleolytic degradation by RNase J, which is absent in E. coli but present in most bacteria lacking RNase E. This pathway, which closely resembles the mechanism by which deadenylated mRNA is degraded in eukaryotic cells, explains the stabilizing influence of 5'-terminal stem-loops in such bacteria.
Assuntos
Bacillus subtilis/genética , Estabilidade de RNA , RNA Bacteriano/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/fisiologia , Fosforilação , Pirofosfatases/genética , Pirofosfatases/fisiologia , Ribonucleases/metabolismo , Ribonucleases/fisiologia , Nudix HidrolasesRESUMO
We previously showed that ribosomes initiating translation of the B. subtilis hbs mRNA at a strong Shine-Dalgarno sequence block the 5' exoribonuclease RNase J1 from degrading into the coding sequence. Here, we identify new and previously unsuspected features of this mRNA. First, we identify RNase Y as the endoribonuclease that cleaves the highly structured 5'-UTR to give access to RNase J1. Cleavage by RNase Y at this site is modulated by a 14-bp long-range interaction between the 5'- and 3-UTRs that partially overlaps the cleavage site. In addition to this maturation/degradation pathway, we discovered a new and ultimately more important RNase Y cleavage site in the very early coding sequence, masked by the initiating ribosome. Thus, two independent pathways compete with ribosomes to tightly link hbs mRNA stability to translation initiation; in one case the initiating ribosome competes directly with RNase J1 and in the other with RNase Y. This is in contrast to prevailing models in Escherichia coli where ribosome traffic over the ORF is the main source of protection from RNases. Indeed, a second RNase Y cleavage site later in the hbs ORF plays no role in its turnover, confirming that for this mRNA at least, initiation is key.
Assuntos
Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Ribonucleases/metabolismo , Ribossomos/metabolismo , Regiões 3' não Traduzidas/genética , Regiões 5' não Traduzidas/genética , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Sequência de Bases , Modelos Genéticos , Biossíntese de Proteínas/genética , Clivagem do RNA , Estabilidade de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribonucleases/genética , Ribossomos/genéticaRESUMO
tRNAs are synthesized as precursor RNAs that have to undergo processing steps to become functional. Yeast Trz1 is a key endoribonuclease involved in the 3Î maturation of tRNAs in all domains of life. It is a member of the ß-lactamase family of RNases, characterized by an HxHxDH sequence motif involved in coordination of catalytic Zn-ions. The RNase Z family consists of two subfamilies: the short (250-400 residues) and the long forms (about double in size). Short form RNase Z enzymes act as homodimers: one subunit embraces tRNA with a protruding arm, while the other provides the catalytic site. The long form is thought to contain two fused ß-lactamase domains within a single polypeptide. Only structures of short form RNase Z enzymes are known. Here we present the 3.1 Å crystal structure of the long-form Trz1 from Saccharomyces cerevisiae. Trz1 is organized into two ß-lactamase domains connected by a long linker. The N-terminal domain has lost its catalytic residues, but retains the long flexible arm that is important for tRNA binding, while it is the other way around in the C-terminal domain. Trz1 likely evolved from a duplication and fusion of the gene encoding the monomeric short form RNase Z.
Assuntos
Endorribonucleases/química , Proteínas de Saccharomyces cerevisiae/química , Sequência de Aminoácidos , Domínio Catalítico , Sequência Conservada , Cristalografia por Raios X , Evolução Molecular , Modelos Moleculares , Fases de Leitura Aberta , Conformação Proteica , Domínios Proteicos , RNA de Transferência/metabolismo , Proteínas Recombinantes de Fusão/química , Saccharomyces cerevisiae/enzimologia , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
RNase III proteins recognize double-stranded RNA structures and catalyze endoribonucleolytic cleavages that often regulate gene expression. Here, we characterize the functions of RNC3 and RNC4, two Arabidopsis thaliana chloroplast Mini-RNase III-like enzymes sharing 75% amino acid sequence identity. Whereas rnc3 and rnc4 null mutants have no visible phenotype, rnc3/rnc4 (rnc3/4) double mutants are slightly smaller and chlorotic compared with the wild type. In Bacillus subtilis, the RNase Mini-III is integral to 23S rRNA maturation. In Arabidopsis, we observed imprecise maturation of 23S rRNA in the rnc3/4 double mutant, suggesting that exoribonucleases generated staggered ends in the absence of specific Mini-III-catalyzed cleavages. A similar phenotype was found at the 3' end of the 16S rRNA, and the primary 4.5S rRNA transcript contained 3' extensions, suggesting that Mini-III catalyzes several processing events of the polycistronic rRNA precursor. The rnc3/4 mutant showed overaccumulation of a noncoding RNA complementary to the 4.5S-5S rRNA intergenic region, and its presence correlated with that of the extended 4.5S rRNA precursor. Finally, we found rnc3/4-specific intron degradation intermediates that are probable substrates for Mini-III and show that B. subtilis Mini-III is also involved in intron regulation. Overall, this study extends our knowledge of the key role of Mini-III in intron and noncoding RNA regulation and provides important insight into plastid rRNA maturation.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Cloroplastos/metabolismo , Íntrons/genética , RNA Ribossômico/genética , Ribonuclease III/metabolismo , Sequência de Aminoácidos , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Bacillus subtilis/metabolismo , Sequência de Bases , Evolução Molecular , Éxons/genética , Teste de Complementação Genética , Modelos Biológicos , Dados de Sequência Molecular , Mutação/genética , Polirribossomos/metabolismo , Estrutura Terciária de Proteína , Estabilidade de RNA , RNA Ribossômico/metabolismo , RNA Ribossômico 23S/genética , RNA não Traduzido/genética , Ribossomos/metabolismo , Análise de Sequência de RNA , Homologia de Sequência de Aminoácidos , TransgenesRESUMO
We recently identified a novel ribonuclease in Bacillus subtilis called Rae1 that cleaves mRNAs in a translation-dependent manner. Rae1 is a member of the NYN/PIN family of ribonucleases and is highly conserved in the Firmicutes, the Cyanobacteria and the chloroplasts of photosynthetic algae and plants. We have proposed a model in which Rae1 enters the A-site of ribosomes that are paused following translation of certain sequences that are still ill-defined. In the only case identified thus far, Rae1 cleaves between a conserved glutamate and lysine codon during translation of a short peptide called S1025. Certain other codons are also tolerated on either side of the cleavage site, but these are recognized less efficiently. The model of Rae1 docked in the A-site allows us to make predictions about which conserved residues may be important for recognition of mRNA, the tRNA in the adjacent P-site and binding to the 50S ribosome subunit.
Assuntos
Bacillus subtilis/enzimologia , Proteínas de Bactérias/metabolismo , Códon , Endonucleases/metabolismo , RNA Bacteriano/metabolismo , Ribossomos/enzimologia , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Endonucleases/genética , RNA Bacteriano/genética , RNA de Transferência/genética , RNA de Transferência/metabolismo , Ribossomos/genéticaRESUMO
Proteomic studies have established that Trz1, Nuc1 and mutarotase form a complex in yeast. Trz1 is a ß-lactamase-type RNase composed of two ß-lactamase-type domains connected by a long linker that is responsible for the endonucleolytic cleavage at the 3'-end of tRNAs during the maturation process (RNase Z activity); Nuc1 is a dimeric mitochondrial nuclease involved in apoptosis, while mutarotase (encoded by YMR099C) catalyzes the conversion between the α- and ß-configuration of glucose-6-phosphate. Using gel filtration, small angle X-ray scattering and electron microscopy, we demonstrated that Trz1, Nuc1 and mutarotase form a very stable heterohexamer, composed of two copies of each of the three subunits. A Nuc1 homodimer is at the center of the complex, creating a two-fold symmetry and interacting with both Trz1 and mutarotase. Enzymatic characterization of the ternary complex revealed that the activities of Trz1 and mutarotase are not affected by complex formation, but that the Nuc1 activity is completely inhibited by mutarotase and partially by Trz1. This suggests that mutarotase and Trz1 might be regulators of the Nuc1 apoptotic nuclease activity.
Assuntos
Carboidratos Epimerases/química , Endonucleases/química , Endorribonucleases/química , Exonucleases/química , Proteínas de Saccharomyces cerevisiae/química , Sequência de Aminoácidos , Carboidratos Epimerases/genética , Endonucleases/genética , Endorribonucleases/genética , Exonucleases/genética , Estabilidade Proteica , Estrutura Secundária de Proteína , Proteínas de Saccharomyces cerevisiae/genética , Espalhamento a Baixo ÂnguloRESUMO
The recent findings that the narrow-specificity endoribonuclease RNase III and the 5' exonuclease RNase J1 are not essential in the Gram-positive model organism,Bacillus subtilis, facilitated a global analysis of internal 5' ends that are generated or acted upon by these enzymes. An RNA-Seq protocol known as PARE (Parallel Analysis of RNA Ends) was used to capture 5' monophosphorylated RNA ends in ribonuclease wild-type and mutant strains. Comparison of PARE peaks in strains with RNase III present or absent showed that, in addition to its well-known role in ribosomal (rRNA) processing, many coding sequences and intergenic regions appeared to be direct targets of RNase III. These target sites were, in most cases, not associated with a known antisense RNA. The PARE analysis also revealed an accumulation of 3'-proximal peaks that correlated with the absence of RNase J1, confirming the importance of RNase J1 in degrading RNA fragments that contain the transcription terminator structure. A significant result from the PARE analysis was the discovery of an endonuclease cleavage just 2 nts downstream of the 16S rRNA 3' end. This latter observation begins to answer, at least for B. subtilis, a long-standing question on the exonucleolytic versus endonucleolytic nature of 16S rRNA maturation.
Assuntos
Bacillus subtilis/genética , Exorribonucleases/metabolismo , RNA Mensageiro/metabolismo , RNA Ribossômico/metabolismo , Ribonuclease III/metabolismo , Sistemas de Transporte de Aminoácidos Neutros/genética , Bacillus subtilis/enzimologia , Bacillus subtilis/metabolismo , Mutação , Óperon , Processamento Pós-Transcricional do RNA , RNA Bacteriano/química , RNA Bacteriano/metabolismo , RNA Mensageiro/química , RNA Ribossômico/química , RNA Ribossômico 16S/metabolismo , RNA Citoplasmático Pequeno/metabolismo , Análise de Sequência de RNARESUMO
RsaE is the only known trans-acting small regulatory RNA (sRNA) besides the ubiquitous 6S RNA that is conserved between the human pathogen Staphylococcus aureus and the soil-dwelling Firmicute Bacillus subtilis. Although a number of RsaE targets are known in S. aureus, neither the environmental signals that lead to its expression nor its physiological role are known. Here we show that expression of the B. subtilis homolog of RsaE is regulated by the presence of nitric oxide (NO) in the cellular milieu. Control of expression by NO is dependent on the ResDE two-component system in B. subtilis and we determined that the same is true in S. aureus. Transcriptome and proteome analyses revealed that many genes with functions related to oxidative stress and oxidation-reduction reactions were up-regulated in a B. subtilis strain lacking this sRNA. We have thus renamed it RoxS. The prediction of RoxS-dependent mRNA targets also suggested a significant enrichment for mRNAs related to respiration and electron transfer. Among the potential direct mRNA targets, we have validated the ppnKB mRNA, encoding an NAD+/NADH kinase, both in vivo and in vitro. RoxS controls both translation initiation and the stability of this transcript, in the latter case via two independent pathways implicating RNase Y and RNase III. Furthermore, RNase Y intervenes at an additional level by processing the 5' end of the RoxS sRNA removing about 20 nucleotides. Processing of RoxS allows it to interact more efficiently with a second target, the sucCD mRNA, encoding succinyl-CoA synthase, thus expanding the repertoire of targets recognized by this sRNA.
Assuntos
Óxido Nítrico/metabolismo , RNA Bacteriano/genética , RNA não Traduzido/genética , Infecções Estafilocócicas/genética , Transcrição Gênica , Bacillus subtilis/genética , Regulação Bacteriana da Expressão Gênica , Homeostase/genética , Óxido Nítrico/genética , Oxirredução , RNA Bacteriano/biossíntese , RNA Mensageiro/genética , RNA não Traduzido/biossíntese , Ribonuclease III/genética , Infecções Estafilocócicas/microbiologia , Infecções Estafilocócicas/patologia , Staphylococcus aureus/genéticaRESUMO
Stable RNA maturation is a key process in the generation of functional RNAs, and failure to correctly process these RNAs can lead to their elimination through quality control mechanisms. Studies of the maturation pathways of ribosomal RNA and transfer RNA in Bacillus subtilis showed they were radically different from Escherichia coli and led to the identification of new B. subtilis-specific enzymes. We noticed that, despite their important roles in translation, a number of B. subtilis small stable RNAs still did not have characterised maturation pathways, notably the tmRNA, involved in ribosome rescue, and the RNase P RNA, involved in tRNA maturation. Here, we show that tmRNA is matured by RNase P and RNase Z at its 5' and 3' extremities, respectively, whereas the RNase P RNA is matured on its 3' side by RNase Y. Recent evidence that several RNases are not essential in B. subtilis prompted us to revisit maturation of the scRNA, a component of the signal recognition particle involved in co-translational insertion of specific proteins into the membrane. We show that RNase Y is also involved in 3' processing of scRNA. Lastly, we identified some of the enzymes involved in the turnover of these three stable RNAs.