Добірка наукової літератури з теми "RNase J2"

RNase J enzymes are metallohydrolases that are involved in RNA maturation and RNA recycling, govern gene expression in bacteria, and catalyze both exonuclease and endonuclease activity. The catalytic activity of RNase J is regulated by multiple mechanisms which include oligomerization, conformational changes to aid substrate recognition, and the metal cofactor at the active site. However, little is known of how RNase J paralogs differ in expression and activity. Here we describe structural and biochemical features of two Staphylococcus epidermidis RNase J paralogs, RNase J1 and RNase J2. RNase J1 is a homodimer with exonuclease activity aided by two metal cofactors at the active site. RNase J2, on the other hand, has endonuclease activity and one metal ion at the active site and is predominantly a monomer. We note that the expression levels of these enzymes vary across Staphylococcal strains. Together, these observations suggest that multiple interacting RNase J paralogs could provide a strategy for functional improvisation utilizing differences in intracellular concentration, quaternary structure, and distinct active site architecture despite overall structural similarity.

The regulation of RNA decay is now widely recognized as having a central role in bacterial adaption to environmental stress. Here we present an overview on the diversity of ribonucleases (RNases) and their impact at the posttranscriptional level in the human pathogenStaphylococcus aureus. RNases in prokaryotes have been mainly studied in the two model organismsEscherichia coliandBacillus subtilis. Based on identified RNases in these two models, putative orthologs have been identified inS. aureus. The main staphylococcal RNases involved in the processing and degradation of the bulk RNA are (i) endonucleases RNase III and RNase Y and (ii) exonucleases RNase J1/J2 and PNPase, having 5′ to 3′ and 3′ to 5′ activities, respectively. The diversity and potential roles of each RNase and of Hfq and RppH are discussed in the context of recent studies, some of which are based on next-generation sequencing technology.

Moonlighting proteins are proteins with more than one function. During the past 25 years, they have been found to be rather widespread in bacteria. In Bacillus subtilis, moonlighting has been disclosed to occur via DNA, protein or RNA binding or protein phosphorylation. In addition, two metabolic enzymes, enolase and phosphofructokinase, were localized in the degradosome-like network (DLN) where they were thought to be scaffolding components. The DLN comprises the major endoribonuclease RNase Y, 3′-5′ exoribonuclease PnpA, endo/5′-3′ exoribonucleases J1/J2 and helicase CshA. We have ascertained that the metabolic enzyme GapA is an additional component of the DLN. In addition, we identified two small proteins that bind scaffolding components of the degradosome: SR1P encoded by the dual-function sRNA SR1 binds GapA, promotes the GapA-RNase J1 interaction and increases the RNase J1 activity. SR7P encoded by the dual-function antisense RNA SR7 binds to enolase thereby enhancing the enzymatic activity of enolase bound RNase Y. We discuss the role of small proteins in modulating the activity of two moonlighting proteins.

ABSTRACT rpsO mRNA, a small monocistronic mRNA that encodes ribosomal protein S15, was used to study aspects of mRNA decay initiation in Bacillus subtilis. Decay of rpsO mRNA in a panel of 3′-to-5′ exoribonuclease mutants was analyzed using a 5′-proximal oligonucleotide probe and a series of oligonucleotide probes that were complementary to overlapping sequences starting at the 3′ end. The results provided strong evidence that endonuclease cleavage in the body of the message, rather than degradation from the native 3′ end, is the rate-determining step for mRNA decay. Subsequent to endonuclease cleavage, the upstream products were degraded by polynucleotide phosphorylase (PNPase), and the downstream products were degraded by the 5′ exonuclease activity of RNase J1. The rpsO mRNA half-life was unchanged in a strain that had decreased RNase J1 activity and no RNase J2 activity, but it was 2.3-fold higher in a strain with decreased activity of RNase Y, a recently discovered RNase of B. subtilis encoded by the ymdA gene. Accumulation of full-length rpsO mRNA and its decay intermediates was analyzed using a construct in which the rpsO transcription unit was under control of a bacitracin-inducible promoter. The results were consistent with RNase Y-mediated initiation of decay. This is the first report of a specific mRNA whose stability is determined by RNase Y.

ABSTRACT The Bacillus subtilis rpsO gene specifies a small (388-nucleotide), monocistronic mRNA that encodes ribosomal protein S15. We showed earlier that rpsO mRNA decay intermediates accumulated to a high level in a strain lacking polynucleotide phosphorylase. Here, we used inducibly expressed derivatives of rpsO, encoding smaller RNAs that had the complex 5′ region deleted, to study aspects of mRNA processing in B. subtilis. An IPTG (isopropyl-β-d-thiogalactopyranoside)-inducible rpsO transcript that contained lac sequences at the 5′ end, called lac-rpsO RNA, was shown to undergo processing to result in an RNA that was 24 nucleotides shorter than full length. Such processing was dependent on the presence of an accessible 5′ terminus; a lac-rpsO RNA that contained a strong stem-loop at the 5′ end was not processed and was extremely stable. Interestingly, this stability depended also on ribosome binding to a nearby Shine-Dalgarno sequence but was independent of downstream translation. Either RNase J1 or RNase J2 was capable of processing lac-rpsO RNA, demonstrating for the first time a particular in vivo processing event that could be catalyzed by both enzymes. Decay intermediates were detected in the pnpA strain only for a lac-rpsO RNA that was untranslated. Analysis of processing of an untranslated lac-rpsO RNA in the pnpA strain shortly after induction of transcription suggested that endonuclease cleavage at 3′-proximal sites was an early step in turnover of mRNA.

ABSTRACTKnowledge of how microorganisms respond and adapt to low-pressure (LP) environments is limited. Previously,Bacillus subtilisstrain WN624 was grown at the near-inhibitory LP of 5 kPa for 1,000 generations and strain WN1106, which exhibited increased relative fitness at 5 kPa, was isolated. Genomic sequence differences between ancestral strain WN624 and LP-evolved strain WN1106 were identified using whole-genome sequencing. LP-evolved strain WN1106 carried amino acid-altering mutations in the coding sequences of only seven genes (fliI,parC,ytoI,bacD,resD,walK, andyvlD) and a single 9-nucleotide in-frame deletion in thernjBgene that encodes RNase J2, a component of the RNA degradosome. By using a collection of frozen stocks of the LP-evolved culture taken at 50-generation intervals, it was determined that (i) the fitness increase at LP occurred rapidly, while (ii) mutation acquisition exhibited complex kinetics. A knockout mutant ofrnjBwas shown to increase the competitive fitness ofB. subtilisat both LP and standard atmospheric pressure.

ABSTRACT Pili in Gram-positive bacteria play a major role in the colonization of host tissue and in the development of biofilms. They are promising candidates for vaccines or drug targets since they are highly immunogenic and share common structural and functional features among various Gram-positive pathogens. Numerous publications have helped build a detailed understanding of pilus surface assembly, yet regulation of pilin gene expression has not been well defined. Utilizing a monoclonal antibody developed against the Enterococcus faecalis major pilus protein EbpC, we identified mutants from a transposon (Tn) insertion library which lack surface-exposed Ebp pili. In addition to insertions in the ebp regulon, an insertion in ef1184 (dapA) significantly reduced levels of EbpC. Analysis of in-frame dapA deletion mutants and mutants with the downstream gene rnjB deleted further demonstrated that rnjB was responsible for the deficiency of EbpC. Sequence analysis revealed that rnjB encodes a putative RNase J2. Subsequent quantitative real-time PCR (qRT-PCR) and Northern blotting demonstrated that the ebpABC mRNA transcript level was significantly decreased in the rnjB deletion mutant. In addition, using a reporter gene assay, we confirmed that rnjB affects the expression of the ebpABC operon. Functionally, the rnjB deletion mutant was attenuated in its ability to produce biofilm, similar to that of an ebpABC deletion mutant which lacks Ebp pili. Together, these results demonstrate the involvement of rnjB in E. faecalis pilin gene expression and provide insight into a novel mechanism of regulation of pilus production in Gram-positive pathogens.

RNA degrading enzymes and multi-enzyme complexes govern a variety of cellular processes. The role of these enzymes and multi-enzyme assemblies has been suggested to govern gene expression levels in bacteria with modulations leading to a so-called phenotypic switch from the persistent (biofilm forming) to a virulent phase. The role of these enzymes in modulating RNA-dependent signal transduction is less understood although several studies implicate these enzymes in regulating the intracellular levels of RNA messengers. Given the multi-functional roles of these enzymes from housekeeping functions such as RNA recycling to mediating bacterial cell phenotypes, understanding these proteins and multi-enzyme complexes is essential to understand bacterial physiology. The work reported in this thesis represents one step in on-going studies to understand the cellular and mechanistic triggers that enable the assembly of multi-enzyme complexes to degrade RNA. Structural and biochemical characterization of individual components provide an insight into the rationale for a multienzyme assembly. These multi-enzyme complexes, also referred to as the degradosome, have been suggested to be context dependent sequestration of RNA modification and degrading enzymes under specific cellular or environmental cues. These enzymes and complexes have also been demonstrated to influence the phenotype in Staphylococci. Biochemical and structural features of three components of this complex viz., PNPase, RNase J1 and RNase J2 are described in this thesis. This thesis is organized as follows: Chapter 1 provides an introduction to RNA metabolism and degradation in prokaryotes. One section of this chapter provides a compilation of literature describing structural and biochemical features of enzymes associated with the degradosome in bacteria. This description is designed to place the lacunae in our understanding of the RNA degradation process alongside the progress achieved in the characterization of individual component enzymes. A brief description of the degradosome complex is also provided to highlight the differences between Gram-positive and Gram-negative bacteria as well as the components in different bacterial species. This is followed by an introduction to the role of RNA mediated processes in Staphylococci to place the work described in this thesis in the broad context of this bacterial pathogen. The emphasis here is on the bacterial phenotype, in particular the virulent phase characterized by secretion of multiple exotoxins. Finally, a section on reported differences in the degradosome components between Staphylococci and other bacteria is compiled to phrase the broad research questions in this area and the aim and scope of the work described in this thesis. The structure and biochemical features of PNPase are described in Chapter 2. Polynucleotide phosphorylase catalyzes both 3'-5' exoribonuclease and polyadenylation reactions. The crystal structure of Staphylococcus epidermidis PNPase revealed a bound phosphate in the PH2 domain of each protomer coordinated by three adjacent serine residues. Mutational analysis revealed that phosphate coordination by these serine residues was essential to maintain the catalytic center in an active conformation. We note that PNPase forms a complex with RNase J1 and RNase J2 without substantially altering either exoribonuclease and polyadenylation activity of this enzyme. This decoupling of catalytic activity from proteinprotein interactions suggests that association of these endo- or exo-ribonucleases with PNPase could be more relevant for cellular localization or concerted targeting of structured RNA for recycling. There are two RNase J paralogues in Staphylococci- RNase J1 and RNase J2. The structural and biochemical features of these two enzymes and the characterization of the interactions between these two RNase J components in vitro is described in Chapter 3. A comparison of these enzymes with previously described RNase J homologs reveals distinct features that provide a potential rationale for the existence of these RNase J paralogs (in most Gram-positive bacteria). Enzyme assays were performed to determine the relative endo- and exoribonuclease activity of RNase J1 and RNase J2. We note that these activities rely on a metal ion at the active site. An analysis of this metal co-factor and its implication for the reaction mechanism is described in this chapter. Structural studies provided another perspective to the role of these enzymes and rationale for association (the RNase J1- RNase J2 complex). While the overall structures of these enzymes are broadly similar, differences in the active site suggest that the two paralogues might adopt different reaction mechanisms. These studies thus suggest a need to revisit a prevailing hypothesis that RNase J is a functional homologue of E. coli RNase E despite poor sequence similarity. Chapter four provides a summary of biochemical information obtained on the three RNA degrading enzymes. These results suggest a link between the regulation of these enzymes and their assembly in vivo. Indeed, this study, as well as other reports in this area, suggest substantial interactions between multi-enzyme RNA-degrading complex and the biochemical pathways that govern energy metabolism. In the case of PNPase, for example, both citrate and ATP influence PNPase activity. While the association between multiple signal transduction pathways and RNA recycling is not surprising, further work to establish conditional correlation between these intracellular networks is essential to understand these mechanisms. The work reported in this thesis also provides a framework for the identification of target mRNA that might bind the multienzyme RNA degradation complex. The structural and biochemical information of all degradosome components (of which three have been described in this thesis) would substantially aid in recreating the assembly of this multi-enzyme complex in vitro