Добірка наукової літератури з теми "RNase J1"

ABSTRACT 23S rRNA maturation in Bacillus subtilis is catalyzed by the recently characterized enzyme Mini-RNase-III. Mini-III is dispensable, however, and 23S rRNA is matured by other ribonucleases in strains lacking this enzyme. Here we show that these RNases are the 5′-to-3′ exoribonuclease RNase J1 and the 3′-to-5′ exoribonucleases, principally RNase PH and YhaM.

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 instability of messenger RNA is crucial to the control of gene expression. In Bacillus subtilis, RNase Y is the major decay-initiating endoribonuclease. Here, we show how this key enzyme regulates its own synthesis by modulating the longevity of its mRNA. Autoregulation is achieved through cleavages in two regions of the rny (RNase Y) transcript: (i) within the first ~100 nucleotides of the open reading frame, immediately inactivating the mRNA for further rounds of translation; (ii) cleavages in the rny 5′ UTR, primarily within the 5′-terminal 50 nucleotides, creating entry sites for the 5′ exonuclease J1 whose progression is blocked around position −15 of the rny mRNA, potentially by initiating ribosomes. This links the functional inactivation of the transcript by RNase J1 to translation efficiency, depending on the ribosome occupancy at the translation initiation site. By these mechanisms, RNase Y can initiate degradation of its own mRNA when the enzyme is not occupied with degradation of other RNAs and thus prevent its overexpression beyond the needs of RNA metabolism.

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.

The involvement of the recently characterized 5′ exonuclease activity of RNase J1 and endonuclease activity of RNase Y in the turnover of ΔermCmRNA inBacillus subtiliswas investigated. Evidence is presented that both of these activities determine the half-life of ΔermCmRNA.

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.

L'élaboration du contrôle de l'expression d'un gène peut s'effectuer par le contrôle de la demi-vie de ce même ARN. Si les processus de maturation et de dégradation de l'ARNm chez Escherichia coli sont à peu près compris à l'heure actuelle, tel n'est pas le cas chez Bacillus subtilis, le paradigme des bactéries à Gram-positif. Ainsi, sur les trente-trois ribonucléases (RNases) identifiées chez ces deux organismes à ce jour, huit seulement leur sont communes. B. Subtilis n'a pas d'homologue des RNases essentielles chez E. Coli, la RNase E et Poligoribonucléase. Deux enzymes paralogues récemment découvertes, les RNases Jl et J2, codées par les gènes rnjA et rnjB, sont de bonnes candidates de B. Subtilis pour jouer un rôle analogue à celui de la RNase E. De façon remarquable, la RNase Jl possède également une activité exoribonucléolytique 5'-3'. L'identification de ces nouvelles enzymes a ouvert une nouvelle voie d'étude des mécanismes de dégradation de l'ARNm chez B. Subtilis. Au cours de cette étude chez B. Subtilis, y ai pu participer à la caractérisation du rôle de la RNase Jl dans la maturation de l'extrémité 5' de TARN ribosomique 16S. J'ai également analysé le rôle de la RNase Jl dans la maturation et la dégradation des ARN messagers. J'ai démontré que l'activité exoribonucléasique 5'-3' de la RNase Jl est la principale activité requise dans ces voies de maturation et de dégradation de l'ARNm hbs. Le gêne hbs de B. Subtilis code pour l'homologue de la protéine « histone-like » HU de E. Coli. J'ai aussi démontré que l'activité endoribonucléolytique de la RNase Jl permet in vitro le clivage d'ARN de façon non spécifique dans les régions simple-brin, ce qui constituerait un nouvel atout dans la détermination des structures secondaires de petits ARN
The control of the half-life of a messenger RNA, in addition to that of its synthesis and/or its translation, is another means by which the cell can control the expression of a gene. Although the processes of maturation and degradation of the mRNA in E. Coli are more or less understood at the present time, this is not the case in Bacillus subtilis, the paradigm of Gram-positive bacteria. B. Subtilis has no counterpart of either RNase E, or of oligoribonuclease, both essential RNases in E. Coli, for example. Two recently discovered enzymes, RNases Jl and J2, coded by the rnjA and rnjB genes, respectively, are good candidates to play a role similar to that of RNase E in B. Subtilis. Remarkably, RNase Jl also has 5' to 3' exoribonucleolytic activity. The identification of these enzymes has made possible detailed study of mRNA dégradation in B. Subtilis. For my thesis studies, I participated in the characterization of the role of RNase Jl in the maturation of the 5' extremity o 16S rRNA. I also studied the role of RNase Jl in the maturation and degradation of messenger RNAs. Indeed the depletion of RNase Jl leads to defects in the maturation and degradation of the hbs mRNA, encoding the B. Subtilis homolog of the histone-like protein HU. I was able to determine that thé 5'-3' exoribonucleolytic activity of RNase Jl was the most important for these pathways of maturation and degradation of hbs mRNA. I also showe4 that the endoribonucleolytic activity of RNase Jl cleaves RNAs nonspecifically in single-stranded regions in vitro and that this activity can be used to determine the secondary structures of small RNAs

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