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1.
Biochim Biophys Acta Gene Regul Mech ; 1863(2): 194489, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31935527

RESUMO

Bacteria exhibit an amazing diversity of mechanisms controlling gene expression to both maintain essential functions and modulate accessory functions in response to environmental cues. Over the years, it has become clear that bacterial regulation of gene expression is still far from fully understood. This review focuses on antisense RNAs (asRNAs), a class of RNA regulators defined by their location in cis and their perfect complementarity with their targets, as opposed to small RNAs (sRNAs) which act in trans with only short regions of complementarity. For a long time, only few functional asRNAs in bacteria were known and were almost exclusively found on mobile genetic elements (MGEs), thus, their importance among the other regulators was underestimated. However, the extensive application of transcriptomic approaches has revealed the ubiquity of asRNAs in bacteria. This review aims to present the landscape of studied asRNAs in bacteria by comparing 67 characterized asRNAs from both Gram-positive and Gram-negative bacteria. First we describe the inherent ambiguity in the existence of asRNAs in bacteria, second, we highlight their diversity and their involvement in all aspects of bacterial life. Finally we compare their location and potential mode of action toward their target between Gram-negative and Gram-positive bacteria and present tendencies and exceptions that could lead to a better understanding of asRNA functions.

2.
Sci Rep ; 9(1): 14054, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31575967

RESUMO

Hfq is a RNA-binding protein that plays a pivotal role in the control of gene expression in bacteria by stabilizing sRNAs and facilitating their pairing with multiple target mRNAs. It has already been shown that Hfq, directly or indirectly, interacts with many proteins: RNase E, Rho, poly(A)polymerase, RNA polymerase… In order to detect more Hfq-related protein-protein interactions we have used two approaches, TAP-tag combined with RNase A treatment to access the role of RNA in these complexes, and protein-protein crosslinking, which freezes protein-protein complexes formed in vivo. In addition, we have performed microscale thermophoresis to evaluate the role of RNA in some of the complexes detected and used far-western blotting to confirm some protein-protein interactions. Taken together, the results show unambiguously a direct interaction between Hfq and EF-Tu. However a very large number of the interactions of proteins with Hfq in E. coli involve RNAs. These RNAs together with the interacting protein, may play an active role in the formation of Hfq-containing complexes with previously unforeseen implications for the riboregulatory functions of Hfq.

3.
Biochimie ; 164: 3-16, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30995539

RESUMO

Prokaryotes encounter constant and often brutal modifications to their environment. In order to survive, they need to maintain fitness, which includes adapting their protein expression patterns. Many factors control gene expression but this review focuses on just one, namely antisense RNAs (asRNAs), a class of non-coding RNAs (ncRNAs) characterized by their location in cis and their perfect complementarity with their targets. asRNAs were considered for a long time to be trivial and only to be found on mobile genetic elements. However, recent advances in methodology have revealed that their abundance and potential activities have been underestimated. This review aims to illustrate the role of asRNA in various physiologically crucial functions in both archaea and bacteria, which can be regrouped in three categories: cell maintenance, horizontal gene transfer and virulence. A literature survey of asRNAs demonstrates the difficulties to characterize and assign a role to asRNAs. With the aim of facilitating this task, we describe recent technological advances that could be of interest to identify new asRNAs and to discover their function.


Assuntos
Archaea , Bactérias , Fenômenos Fisiológicos Bacterianos/genética , Transferência Genética Horizontal/genética , RNA Antissenso , Virulência/genética , Archaea/genética , Archaea/patogenicidade , Archaea/fisiologia , Bactérias/genética , Bactérias/patogenicidade , Regulação da Expressão Gênica em Archaea , Regulação Bacteriana da Expressão Gênica , RNA Antissenso/genética , RNA Antissenso/fisiologia , RNA Arqueal/genética , RNA Arqueal/fisiologia , RNA Bacteriano/genética , RNA Bacteriano/fisiologia
4.
Artigo em Inglês | MEDLINE | ID: mdl-30397102

RESUMO

Post-transcriptional addition of poly(A) tails to the 3' end of RNA is one of the fundamental events controlling the functionality and fate of RNA in all kingdoms of life. Although an enzyme with poly(A)-adding activity was discovered in Escherichia coli more than 50 years ago, its existence and role in prokaryotic RNA metabolism were neglected for many years. As a result, it was not until 1992 that E. coli poly(A) polymerase I was purified to homogeneity and its gene was finally identified. Further work revealed that, similar to its role in surveillance of aberrant nuclear RNAs of eukaryotes, the addition of poly(A) tails often destabilizes prokaryotic RNAs and their decay intermediates, thus facilitating RNA turnover. Moreover, numerous studies carried out over the last three decades have shown that polyadenylation greatly contributes to the control of prokaryotic gene expression by affecting the steady-state level of diverse protein-coding and non-coding transcripts including antisense RNAs involved in plasmid copy number control, expression of toxin-antitoxin systems and bacteriophage development. Here, we review the main findings related to the discovery of polyadenylation in prokaryotes, isolation, and characterization and regulation of bacterial poly(A)-adding activities, and discuss the impact of polyadenylation on prokaryotic mRNA metabolism and gene expression.This article is part of the theme issue '5' and 3' modifications controlling RNA degradation'.


Assuntos
Bactérias/metabolismo , Poli A/metabolismo , Poliadenilação , RNA/metabolismo , Células Procarióticas/metabolismo
5.
Nucleic Acids Res ; 46(9): 4733-4751, 2018 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-29529286

RESUMO

Clostridium difficile, a major human enteropathogen, must cope with foreign DNA invaders and multiple stress factors inside the host. We have recently provided an experimental evidence of defensive function of the C. difficile CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) system important for its survival within phage-rich gut communities. Here, we describe the identification of type I toxin-antitoxin (TA) systems with the first functional antisense RNAs in this pathogen. Through the analysis of deep-sequencing data, we demonstrate the general co-localization with CRISPR arrays for the majority of sequenced C. difficile strains. We provide a detailed characterization of the overlapping convergent transcripts for three selected TA pairs. The toxic nature of small membrane proteins is demonstrated by the growth arrest induced by their overexpression. The co-expression of antisense RNA acting as an antitoxin prevented this growth defect. Co-regulation of CRISPR-Cas and type I TA genes by the general stress response Sigma B and biofilm-related factors further suggests a possible link between these systems with a role in recurrent C. difficile infections. Our results provide the first description of genomic links between CRISPR and type I TA systems within defense islands in line with recently emerged concept of functional coupling of immunity and cell dormancy systems in prokaryotes.


Assuntos
Sistemas CRISPR-Cas , Clostridium difficile/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Sistemas Toxina-Antitoxina/genética , Genoma Bacteriano , Genômica , Estabilidade de RNA , RNA Bacteriano/metabolismo
6.
Nucleic Acids Res ; 45(5): 2746-2756, 2017 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-28426097

RESUMO

Polyadenylation is thought to be involved in the degradation and quality control of bacterial RNAs but relatively few examples have been investigated. We used a combination of 5΄-tagRACE and RNA-seq to analyze the total RNA content from a wild-type strain and from a poly(A)polymerase deleted mutant. A total of 178 transcripts were either up- or down-regulated in the mutant when compared to the wild-type strain. Poly(A)polymerase up-regulates the expression of all genes related to the FliA regulon and several previously unknown transcripts, including numerous transporters. Notable down-regulation of genes in the expression of antigen 43 and components of the type 1 fimbriae was detected. The major consequence of the absence of poly(A)polymerase was the accumulation of numerous sRNAs, antisense transcripts, REP sequences and RNA fragments resulting from the processing of entire transcripts. A new algorithm to analyze the position and composition of post-transcriptional modifications based on the sequence of unencoded 3΄-ends, was developed to identify polyadenylated molecules. Overall our results shed new light on the broad spectrum of action of polyadenylation on gene expression and demonstrate the importance of poly(A) dependent degradation to remove structured RNA fragments.


Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Poliadenilação , Polinucleotídeo Adenililtransferase/metabolismo , RNA Bacteriano/metabolismo , Toxinas Bacterianas/biossíntese , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Genoma Bacteriano , Mutação , Polinucleotídeo Adenililtransferase/genética , RNA Antissenso/metabolismo , RNA Mensageiro/metabolismo , RNA não Traduzido/metabolismo
7.
RNA ; 22(10): 1560-73, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27495318

RESUMO

The rpsO-pnp operon encodes ribosomal protein S15 and polynucleotide phosphorylase, a major 3'-5' exoribonuclease involved in mRNA decay in Escherichia coli The gene for the SraG small RNA is located between the coding regions of the rpsO and pnp genes, and it is transcribed in the opposite direction relative to the two genes. No function has been assigned to SraG. Multiple levels of post-transcriptional regulation have been demonstrated for the rpsO-pnp operon. Here we show that SraG is a new factor affecting pnp expression. SraG overexpression results in a reduction of pnp expression and a destabilization of pnp mRNA; in contrast, inhibition of SraG transcription results in a higher level of the pnp transcript. Furthermore, in vitro experiments indicate that SraG inhibits translation initiation of pnp Together, these observations demonstrate that SraG participates in the post-transcriptional control of pnp by a direct antisense interaction between SraG and PNPase RNAs. Our data reveal a new level of regulation in the expression of this major exoribonuclease.


Assuntos
Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Polirribonucleotídeo Nucleotidiltransferase/genética , RNA Bacteriano/genética , RNA Interferente Pequeno/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Homeostase , Óperon , Polirribonucleotídeo Nucleotidiltransferase/metabolismo , RNA Bacteriano/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo
8.
FEMS Microbiol Lett ; 363(13)2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27190161

RESUMO

Bacterial small RNAs (sRNAs) play essential roles in the post-transcriptional control of gene expression. To improve their detection by conventional microarrays, we designed a custom microarray containing a group of probes targeting known and some putative Escherichia coli sRNAs. To assess its potential in detection of sRNAs, RNA profiling experiments were performed with total RNA extracted from E. coli MG1655 cells exponentially grown in rich (Luria-Bertani) and minimal (M9/glucose) media. We found that many sRNAs could yield reasonably strong and statistically significant signals corresponding to nearly all sRNAs annotated in the EcoCyc database. Besides differential expression of two sRNAs (GcvB and RydB), expression of other sRNAs was less affected by the composition of the growth media. Other examples of the differentially expressed sRNAs were revealed by comparing gene expression of the wild-type strain and its isogenic mutant lacking functional poly(A) polymerase I (pcnB). Further, northern blot analysis was employed to validate these data and to assess the existence of new putative sRNAs. Our results suggest that the use of custom microarrays with improved capacities for detection of sRNAs can offer an attractive opportunity for efficient gene expression profiling of sRNAs and their target mRNAs at the whole transcriptome level.


Assuntos
Escherichia coli/genética , Perfilação da Expressão Gênica , Análise de Sequência com Séries de Oligonucleotídeos , Pequeno RNA não Traduzido , Regulação Bacteriana da Expressão Gênica , Fator Proteico 1 do Hospedeiro/genética , RNA Bacteriano , RNA Mensageiro/genética , Transcriptoma
9.
RNA ; 20(10): 1567-78, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25147238

RESUMO

A gene for the Hfq protein is present in the majority of sequenced bacterial genomes. Its characteristic hexameric ring-like core structure is formed by the highly conserved N-terminal regions. In contrast, the C-terminal forms an extension, which varies in length, lacks homology, and is predicted to be unstructured. In Gram-negative bacteria, Hfq facilitates the pairing of sRNAs with their mRNA target and thus affects gene expression, either positively or negatively, and modulates sRNA degradation. In Gram-positive bacteria, its role is still poorly characterized. Numerous sRNAs have been detected in many Gram-positive bacteria, but it is not yet known whether these sRNAs act in association with Hfq. Compared with all other Hfqs, the C. difficile Hfq exhibits an unusual C-terminal sequence with 75% asparagine and glutamine residues, while the N-terminal core part is more conserved. To gain insight into the functionality of the C. difficile Hfq (Cd-Hfq) protein in processes regulated by sRNAs, we have tested the ability of Cd-Hfq to fulfill the functions of the E. coli Hfq (Ec-Hfq) by examining various functions associated with Hfq in both positive and negative controls of gene expression. We found that Cd-Hfq substitutes for most but not all of the tested functions of the Ec-Hfq protein. We also investigated the role of the C-terminal part of the Hfq proteins. We found that the C-terminal part of both Ec-Hfq and Cd-Hfq is not essential but contributes to some functions of both the E. coli and C. difficile chaperons.


Assuntos
Clostridium difficile/genética , Escherichia coli/genética , Fator Proteico 1 do Hospedeiro/genética , Fator Proteico 1 do Hospedeiro/metabolismo , Biossíntese de Proteínas , RNA Bacteriano/genética , RNA Mensageiro/genética , Clostridium difficile/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Fenótipo , Ligação Proteica , RNA Bacteriano/metabolismo , RNA Mensageiro/metabolismo , beta-Galactosidase/metabolismo
10.
RNA Biol ; 10(4): 602-9, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23392248

RESUMO

Discovered in eukaryotes as a modification essential for mRNA function, polyadenylation was then identified as a means used by all cells to destabilize RNA. In Escherichia coli, most accessible 3' RNA extremities are believed to be potential targets of poly(A) polymerase I. However, some RNAs might be preferentially adenylated. After a short statement of the current knowledge of poly(A) metabolism, we discuss how Hfq could affect recognition and polyadenylation of RNA terminated by Rho-independent terminators. Comparison of RNA terminus leads to the proposal that RNAs harboring 3' terminal features required for Hfq binding are not polyadenylated, whereas those lacking these structural elements can gain the oligo(A) tails that initiate exonucleolytic degradation. We also speculate that Hfq stimulates the synthesis of longer tails that could be used as Hfq-binding sites involved in non-characterized functions of Hfq-dependent sRNAs.


Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/química , Exorribonucleases/química , Exorribonucleases/metabolismo , Fator Proteico 1 do Hospedeiro/química , Poli A/metabolismo , Poliadenilação , Estabilidade de RNA , RNA Mensageiro/metabolismo , Pequeno RNA não Traduzido/química , Proteínas de Ligação a RNA/genética , Proteínas Repressoras/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Exorribonucleases/genética , Regulação Bacteriana da Expressão Gênica , Fator Proteico 1 do Hospedeiro/genética , Fator Proteico 1 do Hospedeiro/metabolismo , Poli A/química , Poli A/genética , RNA Mensageiro/química , RNA Mensageiro/genética , Pequeno RNA não Traduzido/genética , Pequeno RNA não Traduzido/metabolismo , Proteínas de Ligação a RNA/química , Proteínas Repressoras/química , Fator Rho/genética , Alinhamento de Sequência
11.
Biochimie ; 95(2): 410-8, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23123524

RESUMO

Polyadenylation is recognized as part of a surveillance machinery for eliminating defective RNA molecules in eukaryotes and prokaryotes. Escherichia coli strains, deficient in poly(A)polymerase I (PAP I), expressed less flagellin compared to wild-type strains. Because flagellin synthesis is a late step in the flagellar biosynthesis pathway, we assessed the role of PAP I in this cascade and in flagella function. Transcription of flhDC, fliA, and fliC was decreased in the PAP I mutant. These results provide evidence that polyadenylation positively controls the expression of genes belonging to the flagellar biosynthesis pathway and that this effect is mediated through the FlhDC master regulator. However, the downshift in flagella gene expression in the mutant strain did not provoke any noticeable defects in the synthesis of flagella, in biofilm formation and in swimming speed although there was a reduction in motility on soft agar. Our data support an alternative hypothesis that the reduced motility of the mutant resulted from an alteration of the cell membrane composition caused in part by the higher level of GlmS (Glucosamine-6P synthase) which accumulates in the mutant. In agreement with this hypothesis the mutant is more sensitive to hydrophobic agents and antibiotics and in particular to vancomycin. We propose that PAP I participates in the ability of the bacteria to adapt to and survive detrimental conditions by constantly monitoring and adjusting to its environment.


Assuntos
Proteínas de Escherichia coli/genética , Escherichia coli/genética , Flagelos/genética , Flagelina/genética , Regulação Bacteriana da Expressão Gênica , Polinucleotídeo Adenililtransferase/genética , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Flagelos/efeitos dos fármacos , Flagelos/metabolismo , Flagelina/metabolismo , Mutação , Poliadenilação/efeitos dos fármacos , Polinucleotídeo Adenililtransferase/metabolismo , Regiões Promotoras Genéticas , Fator sigma/genética , Fator sigma/metabolismo , Transativadores/genética , Transativadores/metabolismo , Transcrição Genética , Vancomicina/farmacologia
12.
Biochimie ; 94(7): 1544-53, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22370051

RESUMO

In all organisms, RNA-binding proteins participate in modulating all the steps in the life cycle of RNA, including transcription, folding, translation and turnover. In bacteria, RNA-binding proteins may be specific for a few RNA targets (e.g., several ribosomal proteins that recognize both rRNA during ribosome assembly and their own mRNAs when acting as highly specific autogenous repressors) or function as global regulators implicated in numerous regulatory networks. Some RNA-binding proteins combine all these features, and this particularly concerns the ribosomal protein S1 and the Sm-like protein Hfq. S1 is a key mRNA-binding protein in gram-negative bacteria; it recognizes mRNA leaders and provides binding of diverse mRNAs to the ribosome at the initiation step of translation. Moreover, S1 is a highly specific autogenous repressor that is able to distinguish its own mRNA from all the others. Hfq is recognized as a global regulator that facilitates small RNA-mRNA interactions in bacteria; it thereby controls the expression of many mRNAs either positively or negatively. In addition, these two proteins were reported to affect transcription, RNA degradation and other processes. Although they have no sequence specificity, Hfq and S1 preferentially bind A/U-rich single-stranded RNA regions; despite this, they nevertheless carry out very different tasks in the cell. This review is focused on the diversity of functions that can be performed by these abundant RNA-binding bacterial proteins.


Assuntos
Fator Proteico 1 do Hospedeiro/metabolismo , Proteínas Ribossômicas/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Regulação da Expressão Gênica , Fator Proteico 1 do Hospedeiro/química , Humanos , Proteínas Ribossômicas/química
13.
Mol Microbiol ; 83(2): 436-51, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22142150

RESUMO

Polyadenylation is a universal post-transcriptional modification involved in degradation and quality control of bacterial RNAs. In Escherichia coli, it is admitted that any accessible RNA 3' end can be tagged by a poly(A) tail for decay. However, we do not have yet an overall view of the population of polyadenylated molecules. The sampling of polyadenylated RNAs presented here demonstrates that rRNA fragments and tRNA precursors originating from the internal spacer regions of the rrn operons, in particular, rrnB are abundant poly(A) polymerase targets. Focused analysis showed that Glu tRNA precursors originating from the rrnB and rrnG transcripts exhibit long 3' trailers that are primarily removed by PNPase and to a lesser extent by RNase II and poly(A) polymerase. Moreover, 3' trimming by exoribonucleases precedes 5' end maturation by RNase P. Interestingly, characterization of RNA fragments that accumulate in a PNPase deficient strain showed that Glu tRNA precursors still harbouring the 5' leader can be degraded by a 3' to 5' quality control pathway involving poly(A) polymerase. This demonstrates that the surveillance of tRNA maturation described for a defective tRNA also applies to a wild-type tRNA.


Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Escherichia coli/metabolismo , Polinucleotídeo Adenililtransferase/metabolismo , RNA Bacteriano/metabolismo , Aminoacil-RNA de Transferência/metabolismo , Estabilidade de RNA
14.
Biochimie ; 92(10): 1458-61, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20603180

RESUMO

GcvB is a non-coding RNA that regulates oppA mRNA in different bacterial species by binding a GcvB GU-rich region named R1 to oppA mRNA. A secondary putative interaction site (PS1) was identified in this study that is able to form a second nearly perfect 10 base-pair duplex between these two RNAs in Escherichia coli. In this work, we have studied whether the formation of a second interaction site could help stabilize the previously reported GcvB/oppA complex. Several mutations and the full deletion of PS1 were engineered. None of these modifications affected the ability of GcvB to control OppA expression. Therefore the second, putative, interaction site appears to be unnecessary for the regulatory function of GcvB with regard to its oppA target mRNA.


Assuntos
Aminoácido Oxirredutases/genética , Proteínas de Transporte/genética , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Lipoproteínas/genética , RNA Bacteriano/metabolismo , Sítios de Ligação , Mutação , RNA Mensageiro/metabolismo , RNA não Traduzido/metabolismo
15.
BMC Mol Biol ; 11: 17, 2010 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-20167073

RESUMO

BACKGROUND: The bacterial Lsm protein, Hfq, is an RNA chaperone involved in many reactions related to RNA metabolism, such as replication and stability, control of small RNA activity and polyadenylation. Despite this wide spectrum of known functions, the global role of Hfq is almost certainly undervalued; its capacity to bind DNA and to interact with many other proteins are only now beginning to be taken into account. RESULTS: The role of Hfq in the maturation and degradation of the rpsO mRNA of E. coli was investigated in vivo. The data revealed a decrease in rpsO mRNA abundance concomitant to an increase in its stability when Hfq is absent. This indicates that the change in mRNA levels in hfq mutants does not result from its modification of RNA stability. Moreover, a series of independent experiments have revealed that the decrease in mRNA level is not a consequence of a reduction of translation efficiency and that Hfq is not directly implicated in translational control of rpsO expression. Reduced steady-state mRNA levels in the absence of Hfq were also shown for rpsT, rpsB and rpsB-tsf, but not for lpp, pnp or tRNA transcripts. The abundance of chimeric transcripts rpsO-lacZ and rpsB-lacZ, whose expression was driven by rpsO and rpsB promoters, respectively, was also lower in the hfq null-mutants, while the beta-galactosidase yield remained about the same as in the parent wild-type strain. CONCLUSIONS: The data obtained suggest that alteration of rpsO, rpsT and rpsB-tsf transcript levels observed under conditions of Hfq deficiency is not caused by the post-transcriptional events, such as mRNA destabilization or changes in translation control, and may rather result from changes in transcriptional activity. So far, how Hfq affects transcription remains unclear. We propose that one of the likely mechanisms of Hfq-mediated modulation of transcription might operate early in the elongation step, when interaction of Hfq with a nascent transcript would help to overcome transcription pauses and to prevent preliminary transcript release.


Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Fator Proteico 1 do Hospedeiro/metabolismo , RNA Mensageiro/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Mutação , Estabilidade de RNA
16.
Prog Mol Biol Transl Sci ; 85: 137-85, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19215772

RESUMO

In Escherichia coli, RNA degradation is orchestrated by the degradosome with the assistance of complementary pathways and regulatory cofactors described in this chapter. They control the stability of each transcript and regulate the expression of many genes involved in environmental adaptation. The poly(A)-dependent degradation machinery has diverse functions such as the degradation of decay intermediates generated by endoribonucleases, the control of the stability of regulatory non coding RNAs (ncRNAs) and the quality control of stable RNA. The metabolism of poly(A) and mechanism of poly(A)-assisted degradation are beginning to be understood. Regulatory factors, exemplified by RraA and RraB, control the decay rates of subsets of transcripts by binding to RNase E, in contrast to regulatory ncRNAs which, assisted by Hfq, target RNase E to specific transcripts. Destabilization is often consecutive to the translational inactivation of mRNA. However, there are examples where RNA degradation is the primary regulatory step.


Assuntos
Escherichia coli/genética , Escherichia coli/metabolismo , Poli A/metabolismo , Estabilidade de RNA , Sequência de Bases , Meio Ambiente , Regulação Bacteriana da Expressão Gênica , Dados de Sequência Molecular , Poli A/genética , Poliadenilação
17.
RNA ; 15(2): 316-26, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19103951

RESUMO

Polyadenylation is an important factor controlling RNA degradation and RNA quality control mechanisms. In this report we demonstrate for the first time that RNase R has in vivo affinity for polyadenylated RNA and can be a key enzyme involved in poly(A) metabolism. RNase II and PNPase, two major RNA exonucleases present in Escherichia coli, could not account for all the poly(A)-dependent degradation of the rpsO mRNA. RNase II can remove the poly(A) tails but fails to degrade the mRNA as it cannot overcome the RNA termination hairpin, while PNPase plays only a modest role in this degradation. We now demonstrate that in the absence of RNase E, RNase R is the relevant factor in the poly(A)-dependent degradation of the rpsO mRNA. Moreover, we have found that the RNase R inactivation counteracts the extended degradation of this transcript observed in RNase II-deficient cells. Elongated rpsO transcripts harboring increasing poly(A) tails are specifically recognized by RNase R and strongly accumulate in the absence of this exonuclease. The 3' oligo(A) extension may stimulate the binding of RNase R, allowing the complete degradation of the mRNA, as RNase R is not susceptible to RNA secondary structures. Moreover, this regulation is shown to occur despite the presence of PNPase. Similar results were observed with the rpsT mRNA. This report shows that polyadenylation favors in vivo the RNase R-mediated pathways of RNA degradation.


Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Exorribonucleases/metabolismo , Poli A/metabolismo , Estabilidade de RNA , RNA Mensageiro/metabolismo , Proteínas Ribossômicas/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Exorribonucleases/genética , Poliadenilação
18.
Nucleic Acids Res ; 36(8): 2570-80, 2008 May.
Artigo em Inglês | MEDLINE | ID: mdl-18334534

RESUMO

In Escherichia coli the glmS gene encoding glucosamine 6-phosphate (GlcN-6-P) synthase GlmS is feedback regulated by GlcN-6-P in a pathway that involves the small RNA GlmZ. Expression of glmS is activated by the unprocessed form of GlmZ, which accumulates when the intracellular GlcN-6-P concentration decreases. GlmZ stabilizes a glmS transcript that derives from processing. Overexpression of a second sRNA, GlmY, also activates glmS expression in an unknown way. Furthermore, mutations in two genes, yhbJ and pcnB, cause accumulation of full-length GlmZ and thereby activate glmS expression. The function of yhbJ is unknown and pcnB encodes poly(A) polymerase PAP-I known to polyadenylate and destabilize RNAs. Here we show that GlmY acts indirectly in a way that depends on GlmZ. When the intracellular GlcN-6-P concentration decreases, GlmY accumulates and causes in turn accumulation of full-length GlmZ, which finally activates glmS expression. In glmZ mutants, GlmY has no effect on glmS, whereas artificially expressed GlmZ can activate glmS expression also in the absence of GlmY. Furthermore, we show that PAP-I acts at the top of this regulatory pathway by polyadenylating and destabilizing GlmY. In pcnB mutants, GlmY accumulates and induces glmS expression by stabilizing full-length GlmZ. Hence, the data reveal a regulatory cascade composed of two sRNAs, which responds to GlcN-6-P and is controlled by polyadenylation.


Assuntos
Proteínas de Escherichia coli/genética , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/genética , Poliadenilação , RNA não Traduzido/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/biossíntese , Proteínas de Escherichia coli/metabolismo , Glucosamina/análogos & derivados , Glucosamina/metabolismo , Glucose-6-Fosfato/análogos & derivados , Glucose-6-Fosfato/metabolismo , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/biossíntese , Mutação , Polinucleotídeo Adenililtransferase/genética , Estabilidade de RNA , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA
19.
Methods Enzymol ; 447: 161-81, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-19161843

RESUMO

Polyadenylation is a posttranscriptional modification of RNA occurring in prokaryotes, eukaryotes, and organelles. Long poly(A) tails help export eukaryotic mRNAs and promote mRNA stability and translation, whereas the short bacterial tails facilitate RNA decay. The scarcity of polyadenylated RNAs is one of the obstacles for investigators studying bacterial polyadenylation. The two methods described in this chapter were developed to determine how the poly(A) binding protein Hfq affects the polyadenylation of bacterial RNAs. The first is a 3'-RACE protocol specific to oligoadenylated RNA. This method was designed to rapidly collect a large amount of poly(A) containing 3'-terminal sequences to perform statistical analysis. The second method is an RNA sizing protocol to analyze the polyadenylation status of primary transcripts that were not efficiently detected by 3'-RACE. The latter procedure is based on Northern blot analysis of 3'-RNA fragments generated by RNase H. In the presence of a gene-specific methylated chimeric RNA-DNA oligonucleotide, the enzyme is directed to a unique cleavage site. The 3'-RNA fragments, differing by just one nucleotide at their 3'-ends, are then separated in polyacrylamide gels.


Assuntos
Proteínas de Escherichia coli/fisiologia , Fator Proteico 1 do Hospedeiro/fisiologia , Chaperonas Moleculares/fisiologia , Poli A/metabolismo , Sequência de Bases , Northern Blotting , Dados de Sequência Molecular , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Espectrofotometria Ultravioleta
20.
BMC Mol Biol ; 8: 92, 2007 Oct 18.
Artigo em Inglês | MEDLINE | ID: mdl-17949481

RESUMO

BACKGROUND: The bacterial Sm-like protein Hfq is known as an important regulator involved in many reactions of RNA metabolism. A prominent function of Hfq is the stimulation of RNA polyadenylation catalyzed by E. coli poly(A) polymerase I (PAP). As a member of the nucleotidyltransferase superfamily, this enzyme shares a high sequence similarity with an other representative of this family, the tRNA nucleotidyltransferase that synthesizes the 3'-terminal sequence C-C-A to all tRNAs (CCA-adding enzyme). Therefore, it was assumed that Hfq might not only influence the poly(A) polymerase in its specific activity, but also other, similar enzymes like the CCA-adding enzyme. RESULTS: Based on the close evolutionary relation of these two nucleotidyltransferases, it was tested whether Hfq is a specific modulator acting exclusively on PAP or whether it also influences the activity of the CCA-adding enzyme. The obtained data indicate that the reaction catalyzed by this enzyme is substantially accelerated in the presence of Hfq. Furthermore, Hfq binds specifically to tRNA transcripts, which seems to be the prerequisite for the observed effect on CCA-addition. CONCLUSION: The increase of the CCA-addition in the presence of Hfq suggests that this protein acts as a stimulating factor not only for PAP, but also for the CCA-adding enzyme. In both cases, Hfq interacts with RNA substrates, while a direct binding to the corresponding enzymes was not demonstrated up to now (although experimental data indicate a possible interaction of PAP and Hfq). So far, the basic principle of these stimulatory effects is not clear yet. In case of the CCA-adding enzyme, however, the presented data indicate that the complex between Hfq and tRNA substrate might enhance the product release from the enzyme.


Assuntos
Proteínas de Escherichia coli/fisiologia , Fator Proteico 1 do Hospedeiro/fisiologia , RNA Nucleotidiltransferases/metabolismo , Sequência de Bases , Ativação Enzimática , Proteínas de Escherichia coli/metabolismo , Cinética , Polinucleotídeo Adenililtransferase/metabolismo , Ligação Proteica , RNA de Transferência/metabolismo , Especificidade por Substrato
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