RESUMEN
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.
Asunto(s)
Bacillus subtilis , Regulación Bacteriana de la Expresión Génica , División del ARN , Estabilidad del ARN , ARN Bacteriano , Staphylococcus aureus , Staphylococcus aureus/genética , Staphylococcus aureus/enzimología , Bacillus subtilis/genética , Bacillus subtilis/enzimología , Bacillus subtilis/metabolismo , ARN Bacteriano/metabolismo , ARN Bacteriano/genética , Estabilidad del ARN/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Endorribonucleasas/metabolismo , Endorribonucleasas/genética , Conformación de Ácido Nucleico , Secuencia de BasesRESUMEN
Non-coding RNAs (sRNA) play a key role in controlling gene expression in bacteria, typically by base-pairing with ribosome binding sites to block translation. The modification of ribosome traffic along the mRNA generally affects its stability. However, a few cases have been described in bacteria where sRNAs can affect translation without a major impact on mRNA stability. To identify new sRNA targets in Bacillus subtilis potentially belonging to this class of mRNAs, we used pulsed-SILAC (stable isotope labeling by amino acids in cell culture) to label newly synthesized proteins after short expression of the RoxS sRNA, the best characterized sRNA in this bacterium. RoxS sRNA was previously shown to interfere with the expression of genes involved in central metabolism, permitting control of the NAD+/NADH ratio in B. subtilis. In this study, we confirmed most of the known targets of RoxS, showing the efficiency of the method. We further expanded the number of mRNA targets encoding enzymes of the TCA cycle and identified new targets. One of these is YcsA, a tartrate dehydrogenase that uses NAD+ as co-factor, in excellent agreement with the proposed role of RoxS in management of NAD+/NADH ratio in Firmicutes. IMPORTANCE Non-coding RNAs (sRNA) play an important role in bacterial adaptation and virulence. The identification of the most complete set of targets for these regulatory RNAs is key to fully identifying the perimeter of its function(s). Most sRNAs modify both the translation (directly) and mRNA stability (indirectly) of their targets. However, sRNAs can also influence the translation efficiency of the target primarily, with little or no impact on mRNA stability. The characterization of these targets is challenging. We describe here the application of the pulsed SILAC method to identify such targets and obtain the most complete list of targets for a defined sRNA.
Asunto(s)
Bacillus subtilis , ARN Pequeño no Traducido , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , NAD/metabolismo , ARN Pequeño no Traducido/genética , ARN Pequeño no Traducido/metabolismo , ARN Bacteriano/metabolismo , ARN Mensajero/metabolismo , Regulación Bacteriana de la Expresión GénicaRESUMEN
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.
Asunto(s)
Endorribonucleasas , Biosíntesis de Proteínas , Endorribonucleasas/genética , Endorribonucleasas/metabolismo , Sistemas de Lectura Abierta , Ribosomas/genética , Ribosomas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismoRESUMEN
The impact of translation on mRNA stability can be varied, ranging from a protective effect of ribosomes that shield mRNA from RNases to preferentially exposing sites of RNase cleavage. These effects can change depending on whether ribosomes are actively moving along the mRNA or stalled at particular sequences or structures or awaiting charged tRNAs. We recently observed that depleting Bacillus subtilis cells of their tRNA maturation enzymes RNase P and RNase Z led to altered mRNA levels of a number of assembly factors involved in the biogenesis of the 30S ribosomal subunit. Here, we extended this study to other assembly factor and non-assembly factor mRNAs in B. subtilis. We additionally identified multiple transcriptional and translational layers of regulation of the rimM operon mRNA that occur in response to the depletion of functional tRNAs. IMPORTANCE The passage of ribosomes across individual mRNAs during translation can have different effects on their degradation, ranging from a protective effect by shielding from ribonucleases to, in some cases, making the mRNA more vulnerable to RNase action. We recently showed that some mRNAs coding for proteins involved in ribosome assembly were highly sensitive to the availability of functional tRNA. Using strains depleted of the major tRNA processing enzymes RNase P and RNase Z, we expanded this observation to a wider set of mRNAs, including some unrelated to ribosome biogenesis. We characterized the impact of tRNA maturase depletion on the rimM operon mRNA and show that it is highly complex, with multiple levels of transcriptional and posttranscriptional effects coming into play.
RESUMEN
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.
Asunto(s)
Genoma Bacteriano , Operón de ARNr , Escherichia coli/metabolismo , Operón , Ribosomas/genética , Ribosomas/metabolismo , ARN Bacteriano/genética , ARN Ribosómico/genética , ARN Ribosómico/metabolismo , Inestabilidad GenómicaRESUMEN
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.
Asunto(s)
Bacillus subtilis/genética , ARN Bacteriano/metabolismo , ARN Pequeño no Traducido/metabolismo , Bacillus subtilis/metabolismo , Proteínas Bacterianas/metabolismo , Carbono/metabolismo , Aptitud Genética , Proteoma , Procesamiento Postranscripcional del ARN , Estabilidad del ARN , ARN Pequeño no Traducido/biosíntesis , ARN Pequeño no Traducido/genética , ARN Pequeño no Traducido/fisiología , Transcripción GenéticaRESUMEN
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.
Asunto(s)
Bacillus subtilis/enzimología , Endorribonucleasas/química , Endorribonucleasas/metabolismo , Geobacillus stearothermophilus/enzimología , Magnesio/metabolismo , Precursores del ARN/metabolismo , ARN Bicatenario/metabolismo , ARN Ribosómico 5S/metabolismo , Secuencia de Aminoácidos , Endorribonucleasas/genética , Conformación de Ácido Nucleico , Precursores del ARN/genética , ARN Bicatenario/genética , ARN Ribosómico 5S/genética , Ribosomas/genética , Ribosomas/metabolismo , Especificidad por SustratoRESUMEN
Most bacterial ribonucleases (RNases) known to date have been identified in either Escherichia coli or Bacillus subtilis. These two organisms lie on opposite poles of the phylogenetic spectrum, separated by 1-3 billion years of evolution. As a result, the RNA maturation and degradation machineries of these two organisms have little overlap, with each having a distinct set of RNases in addition to a core set of enzymes that is highly conserved across the bacterial spectrum. In this paper, we describe what the functions performed by major RNases in these two bacteria, and how the evolutionary space between them can be described by two opposing gradients of enzymes that fade out and fade in, respectively, as one walks across the phylogenetic tree from E. coli to B. subtilis.
La plupart des ribonucléases (RNases) bactériennes connues à ce jour ont été identifiées chez Escherichia coli ou Bacillus subtilis. Ces deux organismes se trouvent aux pôles opposés du spectre phylogénétique, séparés par 13 milliards d'années d'évolution. Par conséquent, les mécanismes de maturation et de dégradation de l'ARN de ces deux organismes se chevauchent peu, chacun possédant un ensemble distinct de RNases en plus d'un ensemble coeur d'enzymes hautement conservées dans tout le spectre bactérien. Dans cet article, nous décrivons les fonctions remplies par les principales RNases de ces deux bactéries, et comment l'espace évolutif qui les sépare peut être décrit par deux gradients opposés d'enzymes qui disparaissent et apparaissent, respectivement, lorsqu'on parcourt l'arbre phylogénétique de E. coli à B. subtilis.
Asunto(s)
Bacillus subtilis/enzimología , Bacillus subtilis/genética , Escherichia coli/enzimología , Escherichia coli/genética , Ribonucleasas , Escherichia coli/metabolismo , Filogenia , Ribonucleasas/química , Ribonucleasas/genética , Ribonucleasas/metabolismoRESUMEN
Ribonucleases can cleave RNAs internally in endoribonucleolytic mode or remove one nucleotide at a time from either the 5' or 3' end through exoribonuclease action. To show direct implication of an RNase in a specific pathway of RNA maturation or decay requires the setting up of in vitro assays with purified enzymes and substrates. This chapter complements Chapter 24 on assays of ribonuclease action in vivo by providing detailed protocols for the assay of B. subtilis RNases with prepared substrates in vitro.
Asunto(s)
Pruebas de Enzimas/métodos , Sondas ARN/metabolismo , ARN Bacteriano/metabolismo , Ribonucleasas/metabolismo , Bacillus subtilis/enzimología , Regulación Bacteriana de la Expresión Génica , CinéticaRESUMEN
Ribonucleases remodel RNAs to render them functional or to send them on their way toward degradation. In our laboratory, we study these pathways in detail using a plethora of different techniques. These can range from the isolation of RNAs in various RNase mutants to determine their implication in maturation or decay pathways by Northern blot, to proving their direct roles in RNA cleavage reactions using purified enzymes and transcribed substrates in vitro. In this chapter, we provide in-depth protocols for the techniques we use daily in the laboratory to assay RNase activity in vivo, with detailed notes on how to get these methods to work optimally. This chapter complements Chapter 25 on assays of ribonuclease action in vitro.
Asunto(s)
Bacillus subtilis/enzimología , Pruebas de Enzimas/métodos , Hibridación in Situ/métodos , ARN Bacteriano/metabolismo , Ribonucleasas/metabolismo , Regulación Bacteriana de la Expresión Génica , CinéticaRESUMEN
Toxin-antitoxin systems are widespread stress-responsive elements, many of whose functions remain largely unknown. Here, we characterize the four DUF1814-family nucleotidyltransferase-like toxins (MenT1-4) encoded by the human pathogen Mycobacterium tuberculosis. Toxin MenT3 inhibited growth of M. tuberculosis when not antagonized by its cognate antitoxin, MenA3. We solved the structures of toxins MenT3 and MenT4 to 1.6 and 1.2 Å resolution, respectively, and identified the biochemical activity and target of MenT3. MenT3 blocked in vitro protein expression and prevented tRNA charging in vivo. MenT3 added pyrimidines (C or U) to the 3'-CCA acceptor stems of uncharged tRNAs and exhibited strong substrate specificity in vitro, preferentially targeting tRNASer from among the 45 M. tuberculosis tRNAs. Our study identifies a previously unknown mechanism that expands the range of enzymatic activities used by bacterial toxins, uncovering a new way to block protein synthesis and potentially treat tuberculosis and other infections.
Asunto(s)
Mycobacterium tuberculosis , Sistemas Toxina-Antitoxina , Tuberculosis , Proteínas Bacterianas/metabolismo , Humanos , Mycobacterium tuberculosis/genética , Nucleotidiltransferasas/metabolismo , ARN de Transferencia/metabolismo , Sistemas Toxina-Antitoxina/genéticaRESUMEN
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.
Asunto(s)
Bacillus subtilis/enzimología , Proteínas Bacterianas/química , Precursores del ARN/metabolismo , Ribonucleasas/química , Subunidades Ribosómicas Grandes Bacterianas/metabolismo , Bacillus subtilis/ultraestructura , Proteínas Bacterianas/ultraestructura , Secuencia de Bases , Microscopía por Crioelectrón , Modelos Moleculares , Precursores del ARN/ultraestructura , Ribonucleasas/ultraestructura , Subunidades Ribosómicas Grandes Bacterianas/ultraestructura , Especificidad por SustratoRESUMEN
Bacterial pathogens often employ RNA regulatory elements located in the 5' untranslated regions (UTRs) to control gene expression. Using a comparative structural analysis, we examine the structure of 5' UTRs at a global scale in the pathogenic bacterium Listeria monocytogenes under different conditions. In addition to discovering an RNA thermoswitch and detecting simultaneous interaction of ribosomes and small RNAs with mRNA, we identify structural changes in the 5' UTR of an mRNA encoding the post-translocation chaperone PrsA2 during infection conditions. We demonstrate that the 5' UTR of the prsA2 mRNA base pairs with the 3' UTR of the full-length hly mRNA encoding listeriolysin O, thus preventing RNase J1-mediated degradation of the prsA2 transcript. Mutants lacking the hly-prsA2 interaction exhibit reduced virulence properties. This work highlights an additional level of RNA regulation, where the mRNA encoding a chaperone is stabilized by the mRNA encoding its substrate.
Asunto(s)
Regulación Bacteriana de la Expresión Génica , Listeria monocytogenes/genética , Listeria monocytogenes/patogenicidad , Chaperonas Moleculares/metabolismo , Factores de Virulencia/genética , Regiones no Traducidas 3'/genética , Regiones no Traducidas 5'/genética , Secuencia de Bases , Proteínas y Péptidos de Choque por Frío/metabolismo , Biblioteca de Genes , Modelos Biológicos , Isomerasa de Peptidilprolil/metabolismo , Estabilidad del ARN/genética , ARN Bacteriano/química , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ribonucleasas/metabolismo , Ribosomas/metabolismo , Temperatura , Virulencia/genética , Factores de Virulencia/metabolismoRESUMEN
Messenger RNA processing and decay is a key mechanism to control gene expression at the post-transcriptional level in response to ever-changing environmental conditions. In this review chapter, we discuss the main ribonucleases involved in these processes in bacteria, with a particular but non-exclusive emphasis on the two best-studied paradigms of Gram-negative and Gram-positive bacteria, E. coli and B. subtilis, respectively. We provide examples of how the activity and specificity of these enzymes can be modulated at the protein level, by co-factor binding and by post-translational modifications, and how they can be influenced by specific properties of their mRNA substrates, such as 5' protective 'caps', nucleotide modifications, secondary structures and translation. This article is part of a Special Issue entitled: RNA and gene control in bacteria edited by Dr. M. Guillier and F. Repoila.
Asunto(s)
Regulación Bacteriana de la Expresión Génica , Procesamiento Postranscripcional del ARN , ARN Bacteriano/metabolismo , ARN Mensajero/metabolismo , Proteínas Bacterianas/metabolismo , Endorribonucleasas/metabolismo , ARN Bacteriano/genética , ARN Mensajero/genéticaRESUMEN
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.
Asunto(s)
Bacillus subtilis/enzimología , Exorribonucleasas/metabolismo , ARN Mensajero/metabolismo , Bacillus subtilis/genética , Proteínas Bacterianas/metabolismo , ARN Polimerasas Dirigidas por ADN/metabolismo , Regulación Bacteriana de la Expresión Génica , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , ARN Mensajero/genética , Transcripción GenéticaRESUMEN
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.
Asunto(s)
Bacillus subtilis/genética , Regulación Bacteriana de la Expresión Génica , Guanosina Pentafosfato/biosíntesis , Iniciación de la Cadena Peptídica Traduccional , ARN Ribosómico 16S/genética , ARN de Transferencia/genética , Bacillus subtilis/metabolismo , Secuencia de Bases , Guanosina Pentafosfato/genética , Guanosina Trifosfato/metabolismo , Ligasas/genética , Ligasas/metabolismo , Conformación de Ácido Nucleico , ARN Ribosómico 16S/química , ARN Ribosómico 16S/metabolismo , ARN de Transferencia/química , ARN de Transferencia/metabolismo , Subunidades Ribosómicas Grandes Bacterianas/genética , Subunidades Ribosómicas Grandes Bacterianas/metabolismo , Subunidades Ribosómicas Pequeñas Bacterianas/genética , Subunidades Ribosómicas Pequeñas Bacterianas/metabolismoRESUMEN
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.
Asunto(s)
Bacillus subtilis/genética , Proteínas Bacterianas/genética , Exorribonucleasas/genética , Eliminación de Gen , Procesamiento de Término de ARN 3' , ARN Ribosómico 16S/genética , Secuencia de Aminoácidos , Bacillus subtilis/metabolismo , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Exorribonucleasas/metabolismo , Metaloproteínas/genética , Metaloproteínas/metabolismo , Precursores del ARN/genética , Precursores del ARN/metabolismo , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , ARN Ribosómico 16S/metabolismo , Homología de Secuencia de Aminoácido , Especificidad de la EspecieRESUMEN
RNases are key enzymes involved in RNA maturation and degradation. Although they play a crucial role in all domains of life, bacteria, archaea, and eukaryotes have evolved with their own sets of RNases and proteins modulating their activities. In bacteria, these enzymes allow modulation of gene expression to adapt to rapidly changing environments. Today, >20 RNases have been identified in both Escherichia coli and Bacillus subtilis, the paradigms of the Gram-negative and Gram-positive bacteria, respectively. However, only a handful of these enzymes are common to these two organisms and some of them are essential to only one. Moreover, although sets of RNases can be very similar in closely related bacteria such as the Firmicutes Staphylococcus aureus and B. subtilis, the relative importance of individual enzymes in posttranscriptional regulation in these organisms varies. In this review, we detail the role of the main RNases involved in RNA maturation and degradation in Gram-positive bacteria, with an emphasis on the roles of RNase J1, RNase III, and RNase Y. We also discuss how other proteins such as helicases can modulate the RNA-degradation activities of these enzymes.
Asunto(s)
ADN Helicasas/fisiología , Bacterias Grampositivas/enzimología , Ribonucleasas/fisiología , Proteínas Bacterianas , Endorribonucleasas/fisiología , Regulación Bacteriana de la Expresión Génica , Bacterias Grampositivas/genética , Estabilidad del ARN , Ribonucleasas/clasificación , Especificidad por SustratoRESUMEN
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.