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1.
Antimicrob Agents Chemother ; : AAC0086421, 2021 Oct 04.
Artigo em Inglês | MEDLINE | ID: mdl-34606341

RESUMO

Rifamycins, such as rifampicin, are potent inhibitors of bacterial RNA polymerases and widely used antibiotics. Usually rifamycin-resistance is associated with mutations in RNAP that preclude rifamycins binding. However, some bacteria have ADP-ribosyl transferases Arr that ADP-ribosylate rifamycin molecules, thus inactivating their antimicrobial activity. Here we directly show that ADP-ribosylation abolishes inhibition of transcription by rifampicin, the most widely used rifamycin antibiotic. We also show that natural rifamycin, Kanglemycin A, which has a unique sugar moiety at the ansa-chain close to the Arr-modification site, does not bind to Arr from M. smegmatis, and thus is not susceptible to inactivation. We, however, found that Kanglemycin A can still be ADP-ribosylated by Arr of an emerging pathogen M. abscessus. Interestingly, the only part of Arr which exhibits no homology between the species is the part that sterically clashes with sugar moiety of Kanglemycin A in M. smegmatis Arr. This suggests that M. abscessus has encountered KglA or rifamycin with similar sugar modification in the course of evolution. The results show that KglA could be effective antimicrobial against some of the Arr encoding bacteria.

2.
Cell Rep ; 36(10): 109671, 2021 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-34496258

RESUMO

Phosphorylation of the RNA polymerase II C-terminal domain Y1S2P3T4S5P6S7 consensus sequence coordinates key events during transcription, and its deregulation leads to defects in transcription and RNA processing. Here, we report that the histone deacetylase activity of the fission yeast Hos2/Set3 complex plays an important role in suppressing cryptic initiation of antisense transcription when RNA polymerase II phosphorylation is dysregulated due to the loss of Ssu72 phosphatase. Interestingly, although single Hos2 and Set3 mutants have little effect, loss of Hos2 or Set3 combined with ssu72Δ results in a synergistic increase in antisense transcription globally and correlates with elevated sensitivity to genotoxic agents. We demonstrate a key role for the Ssu72/Hos2/Set3 mechanism in the suppression of cryptic antisense transcription at the 3' end of convergent genes that are most susceptible to these defects, ensuring the fidelity of gene expression within dense genomes of simple eukaryotes.

3.
Nucleic Acids Res ; 49(15): 8777-8784, 2021 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-34365509

RESUMO

Transcribing RNA polymerase (RNAP) can fall into backtracking, phenomenon when the 3' end of the transcript disengages from the template DNA. Backtracking is caused by sequences of the nucleic acids or by misincorporation of erroneous nucleotides. To resume productive elongation backtracked complexes have to be resolved through hydrolysis of RNA. There is currently no consensus on the mechanism of catalysis of this reaction by Escherichia coli RNAP. Here we used Salinamide A, that we found inhibits RNAP catalytic domain Trigger Loop (TL), to show that the TL is required for RNA cleavage during proofreading of misincorporation events but plays little role during cleavage in sequence-dependent backtracked complexes. Results reveal that backtracking caused by misincorporation is distinct from sequence-dependent backtracking, resulting in different conformations of the 3' end of RNA within the active center. We show that the TL is required to transfer the 3' end of misincorporated transcript from cleavage-inefficient 'misincorporation site' into the cleavage-efficient 'backtracked site', where hydrolysis takes place via transcript-assisted catalysis and is largely independent of the TL. These findings resolve the controversy surrounding mechanism of RNA hydrolysis by E. coli RNA polymerase and indicate that the TL role in RNA cleavage has diverged among bacteria.


Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , RNA Mensageiro/metabolismo , Elongação da Transcrição Genética , Domínio Catalítico , RNA Polimerases Dirigidas por DNA/antagonistas & inibidores , RNA Polimerases Dirigidas por DNA/química , Depsipeptídeos/química , Depsipeptídeos/farmacologia , Escherichia coli/enzimologia , Escherichia coli/genética , Hidrólise , Clivagem do RNA
4.
Nucleic Acids Res ; 48(14): 7914-7923, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32652039

RESUMO

Bacterial RNA polymerase is a potent target for antibiotics, which utilize a plethora of different modes of action, some of which are still not fully understood. Ureidothiophene (Urd) was found in a screen of a library of chemical compounds for ability to inhibit bacterial transcription. The mechanism of Urd action is not known. Here, we show that Urd inhibits transcription at the early stage of closed complex formation by blocking interaction of RNA polymerase with the promoter -10 element, while not affecting interactions with -35 element or steps of transcription after promoter closed complex formation. We show that mutation in the region 1.2 of initiation factor σ decreases sensitivity to Urd. The results suggest that Urd may directly target σ region 1.2, which allosterically controls the recognition of -10 element by σ region 2. Alternatively, Urd may block conformational changes of the holoenzyme required for engagement with -10 promoter element, although by a mechanism distinct from that of antibiotic fidaxomycin (lipiarmycin). The results suggest a new mode of transcription inhibition involving the regulatory domain of σ subunit, and potentially pinpoint a novel target for development of new antibacterials.


Assuntos
Antibacterianos/farmacologia , RNA Polimerases Dirigidas por DNA/antagonistas & inibidores , Regiões Promotoras Genéticas , Tiofenos/farmacologia , Iniciação da Transcrição Genética/efeitos dos fármacos , Antibacterianos/química , Bactérias/enzimologia , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Fator sigma/antagonistas & inibidores , Fator sigma/química , Tiofenos/química
5.
PLoS Pathog ; 16(7): e1008672, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32706832

RESUMO

Most clinical MRSA (methicillin-resistant S. aureus) isolates exhibit low-level ß-lactam resistance (oxacillin MIC 2-4 µg/ml) due to the acquisition of a novel penicillin binding protein (PBP2A), encoded by mecA. However, strains can evolve high-level resistance (oxacillin MIC ≥256 µg/ml) by an unknown mechanism. Here we have developed a robust system to explore the basis of the evolution of high-level resistance by inserting mecA into the chromosome of the methicillin-sensitive S. aureus SH1000. Low-level mecA-dependent oxacillin resistance was associated with increased expression of anaerobic respiratory and fermentative genes. High-level resistant derivatives had acquired mutations in either rpoB (RNA polymerase subunit ß) or rpoC (RNA polymerase subunit ß') and these mutations were shown to be responsible for the observed resistance phenotype. Analysis of rpoB and rpoC mutants revealed decreased growth rates in the absence of antibiotic, and alterations to, transcription elongation. The rpoB and rpoC mutations resulted in decreased expression to parental levels, of anaerobic respiratory and fermentative genes and specific upregulation of 11 genes including mecA. There was however no direct correlation between resistance and the amount of PBP2A. A mutational analysis of the differentially expressed genes revealed that a member of the S. aureus Type VII secretion system is required for high level resistance. Interestingly, the genomes of two of the high level resistant evolved strains also contained missense mutations in this same locus. Finally, the set of genetically matched strains revealed that high level antibiotic resistance does not incur a significant fitness cost during pathogenesis. Our analysis demonstrates the complex interplay between antibiotic resistance mechanisms and core cell physiology, providing new insight into how such important resistance properties evolve.


Assuntos
Proteínas de Bactérias/genética , RNA Polimerases Dirigidas por DNA/genética , Regulação Bacteriana da Expressão Gênica/genética , Staphylococcus aureus Resistente à Meticilina/genética , Proteínas de Ligação às Penicilinas/genética , Resistência beta-Lactâmica/genética , Antibacterianos/farmacologia , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos
6.
Proc Natl Acad Sci U S A ; 117(15): 8462-8467, 2020 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-32238560

RESUMO

In bacteria, the first two steps of gene expression-transcription and translation-are spatially and temporally coupled. Uncoupling may lead to the arrest of transcription through RNA polymerase backtracking, which interferes with replication forks, leading to DNA double-stranded breaks and genomic instability. How transcription-translation coupling mitigates these conflicts is unknown. Here we show that, unlike replication, translation is not inhibited by arrested transcription elongation complexes. Instead, the translating ribosome actively pushes RNA polymerase out of the backtracked state, thereby reactivating transcription. We show that the distance between the two machineries upon their contact on mRNA is smaller than previously thought, suggesting intimate interactions between them. However, this does not lead to the formation of a stable functional complex between the enzymes, as was once proposed. Our results reveal an active, energy-driven mechanism that reactivates backtracked elongation complexes and thus helps suppress their interference with replication.


Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/enzimologia , RNA Mensageiro/metabolismo , Ribossomos/metabolismo , Transcrição Genética , Fatores de Elongação da Transcrição/metabolismo , Sequência de Bases , RNA Polimerases Dirigidas por DNA/genética , Escherichia coli/genética , RNA Mensageiro/genética , Ribossomos/genética , Fatores de Elongação da Transcrição/genética
7.
EcoSal Plus ; 9(1)2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32342856

RESUMO

RNA polymerases (RNAPs) accomplish the first step of gene expression in all living organisms. However, the sequence divergence between bacterial and human RNAPs makes the bacterial RNAP a promising target for antibiotic development. The most clinically important and extensively studied class of antibiotics known to inhibit bacterial RNAP are the rifamycins. For example, rifamycins are a vital element of the current combination therapy for treatment of tuberculosis. Here, we provide an overview of the history of the discovery of rifamycins, their mechanisms of action, the mechanisms of bacterial resistance against them, and progress in their further development.

8.
Mol Cell ; 72(2): 263-274.e5, 2018 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-30244835

RESUMO

Antibiotic-resistant bacterial pathogens pose an urgent healthcare threat, prompting a demand for new medicines. We report the mode of action of the natural ansamycin antibiotic kanglemycin A (KglA). KglA binds bacterial RNA polymerase at the rifampicin-binding pocket but maintains potency against RNA polymerases containing rifampicin-resistant mutations. KglA has antibiotic activity against rifampicin-resistant Gram-positive bacteria and multidrug-resistant Mycobacterium tuberculosis (MDR-M. tuberculosis). The X-ray crystal structures of KglA with the Escherichia coli RNA polymerase holoenzyme and Thermus thermophilus RNA polymerase-promoter complex reveal an altered-compared with rifampicin-conformation of KglA within the rifampicin-binding pocket. Unique deoxysugar and succinate ansa bridge substituents make additional contacts with a separate, hydrophobic pocket of RNA polymerase and preclude the formation of initial dinucleotides, respectively. Previous ansa-chain modifications in the rifamycin series have proven unsuccessful. Thus, KglA represents a key starting point for the development of a new class of ansa-chain derivatized ansamycins to tackle rifampicin resistance.


Assuntos
Produtos Biológicos/farmacologia , Farmacorresistência Bacteriana/efeitos dos fármacos , Mycobacterium tuberculosis/efeitos dos fármacos , Rifabutina/farmacologia , Rifampina/farmacologia , Rifamicinas/farmacologia , Antituberculosos/farmacologia , RNA Polimerases Dirigidas por DNA/genética , Farmacorresistência Bacteriana/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Humanos , Testes de Sensibilidade Microbiana/métodos , Mutação/efeitos dos fármacos , Mutação/genética , Mycobacterium tuberculosis/genética , Thermus thermophilus/efeitos dos fármacos , Thermus thermophilus/genética
9.
Sci Adv ; 4(3): eaap9714, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29546243

RESUMO

Bacterial protein synthesis is intricately connected to metabolic rate. One of the ways in which bacteria respond to environmental stress is through posttranslational modifications of translation factors. Translation elongation factor Tu (EF-Tu) is methylated and phosphorylated in response to nutrient starvation upon entering stationary phase, and its phosphorylation is a crucial step in the pathway toward sporulation. We analyze how phosphorylation leads to inactivation of Escherichia coli EF-Tu. We provide structural and biophysical evidence that phosphorylation of EF-Tu at T382 acts as an efficient switch that turns off protein synthesis by decoupling nucleotide binding from the EF-Tu conformational cycle. Direct modifications of the EF-Tu switch I region or modifications in other regions stabilizing the ß-hairpin state of switch I result in an effective allosteric trap that restricts the normal dynamics of EF-Tu and enables the evasion of the control exerted by nucleotides on G proteins. These results highlight stabilization of a phosphorylation-induced conformational trap as an essential mechanism for phosphoregulation of bacterial translation and metabolism. We propose that this mechanism may lead to the multisite phosphorylation state observed during dormancy and stationary phase.


Assuntos
Fator Tu de Elongação de Peptídeos/química , Fator Tu de Elongação de Peptídeos/metabolismo , Biossíntese de Proteínas , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Guanosina Difosfato/metabolismo , Modelos Moleculares , Nucleotídeos/metabolismo , Fosforilação , Fosfotreonina/metabolismo , Ligação Proteica , Conformação Proteica , Termodinâmica
10.
Curr Opin Microbiol ; 42: 13-18, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-28968546

RESUMO

Accuracy of transcription is essential for productive gene expression, and the past decade has brought new understanding of the mechanisms ensuring transcription fidelity. The discovery of a new catalytic domain, the Trigger Loop, revealed that RNA polymerase can actively choose the correct substrates. Also, the intrinsic proofreading activity was found to proceed via a ribozyme-like mechanism, whereby the erroneous nucleoside triphosphate (NTP) helps its own excision. Factor-assisted proofreading was shown to proceed through an exchange of active centres, a unique phenomenon among proteinaceous enzymes. Furthermore, most recent in vivo studies have revised the roles of transcription accuracy and proofreading factors, as not only required for production of errorless RNAs, but also for prevention of frequent misincorporation-induced pausing that may cause conflicts with fellow RNA polymerases and the replication machinery.


Assuntos
RNA/genética , Transcrição Genética , Domínio Catalítico/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Modelos Moleculares
11.
Nat Commun ; 8: 15774, 2017 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-28585540

RESUMO

Transcription in all living organisms is accomplished by multi-subunit RNA polymerases (msRNAPs). msRNAPs are highly conserved in evolution and invariably share a ∼400 kDa five-subunit catalytic core. Here we characterize a hypothetical ∼100 kDa single-chain protein, YonO, encoded by the SPß prophage of Bacillus subtilis. YonO shares very distant homology with msRNAPs, but no homology with single-subunit polymerases. We show that despite homology to only a few amino acids of msRNAP, and the absence of most of the conserved domains, YonO is a highly processive DNA-dependent RNA polymerase. We demonstrate that YonO is a bona fide RNAP of the SPß bacteriophage that specifically transcribes its late genes, and thus represents a novel type of bacteriophage RNAPs. YonO and related proteins present in various bacteria and bacteriophages have diverged from msRNAPs before the Last Universal Common Ancestor, and, thus, may resemble the single-subunit ancestor of all msRNAPs.


Assuntos
RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Prófagos/enzimologia , Bacillus subtilis/genética , Bacillus subtilis/virologia , Perfilação da Expressão Gênica , Regulação Viral da Expressão Gênica , Prófagos/genética , Proteínas Virais/genética , Proteínas Virais/metabolismo
12.
J Nat Prod ; 80(5): 1558-1562, 2017 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-28398740

RESUMO

The madurastatins are pentapeptide siderophores originally described as containing an unusual salicylate-capped N-terminal aziridine ring. Isolation of madurastatin C1 (1) (also designated MBJ-0034), from Actinomadura sp. DEM31376 (itself isolated from a deep sea sediment), prompted structural reevaluation of the madurastatin siderophores, in line with the recent work of Thorson and Shaaban. NMR spectroscopy in combination with partial synthesis allowed confirmation of the structure of madurastatin C1 (1) as containing an N-terminal 2-(2-hydroxyphenyl)oxazoline in place of the originally postulated aziridine, while absolute stereochemistry was determined via Harada's advanced Marfey's method. Therefore, this work further supports Thorson and Shaaban's proposed structural revision of the madurastatin class of siderophores (madurastatins A1 (2), B1 (3), C1 (1), and MBJ-0036 (4)) as N-terminal 2-(2-hydroxyphenyl)oxazolines.


Assuntos
Aziridinas/química , Oligopeptídeos/química , Peptídeos/química , Piperidonas/química , Sideróforos/química , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Estereoisomerismo
13.
Methods ; 120: 76-84, 2017 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-28434904

RESUMO

The identification of the protein-coding regions of a genome is straightforward due to the universality of start and stop codons. However, the boundaries of the transcribed regions, conditional operon structures, non-coding RNAs and the dynamics of transcription, such as pausing of elongation, are non-trivial to identify, even in the comparatively simple genomes of prokaryotes. Traditional methods for the study of these areas, such as tiling arrays, are noisy, labour-intensive and lack the resolution required for densely-packed bacterial genomes. Recently, deep sequencing has become increasingly popular for the study of the transcriptome due to its lower costs, higher accuracy and single nucleotide resolution. These methods have revolutionised our understanding of prokaryotic transcriptional dynamics. Here, we review the deep sequencing and data analysis techniques that are available for the study of transcription in prokaryotes, and discuss the bioinformatic considerations of these analyses.


Assuntos
Perfilação da Expressão Gênica/métodos , Genoma Bacteriano/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , RNA Bacteriano/genética , Análise de Sequência de RNA/métodos , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Perfilação da Expressão Gênica/instrumentação , Sequenciamento de Nucleotídeos em Larga Escala/instrumentação , Fases de Leitura Aberta/genética , Óperon/genética , Células Procarióticas/química , Células Procarióticas/enzimologia , Células Procarióticas/metabolismo , RNA Bacteriano/isolamento & purificação , Análise de Sequência de RNA/instrumentação , Regiões Terminadoras Genéticas/genética , Sítio de Iniciação de Transcrição , Transcrição Genética , Transcriptoma/genética
14.
Nucleic Acids Res ; 45(3): 1105-1113, 2017 02 17.
Artigo em Inglês | MEDLINE | ID: mdl-28180286

RESUMO

The transcription error rate estimated from mistakes in end product RNAs is 10−3­10−5. We analyzed the fidelity of nascent RNAs from all actively transcribing elongation complexes (ECs) in Escherichia coli and Saccharomyces cerevisiae and found that 1­3% of all ECs in wild-type cells, and 5­7% of all ECs in cells lacking proofreading factors are, in fact, misincorporated complexes. With the exception of a number of sequence-dependent hotspots, most misincorporations are distributed relatively randomly. Misincorporation at hotspots does not appear to be stimulated by pausing. Since misincorporation leads to a strong pause of transcription due to backtracking, our findings indicate that misincorporation could be a major source of transcriptional pausing and lead to conflicts with other RNA polymerases and replication in bacteria and eukaryotes. This observation implies that physical resolution of misincorporated complexes may be the main function of the proofreading factors Gre and TFIIS. Although misincorporation mechanisms between bacteria and eukaryotes appear to be conserved, the results suggest the existence of a bacteria-specific mechanism(s) for reducing misincorporation in protein-coding regions. The links between transcription fidelity, human disease, and phenotypic variability in genetically-identical cells can be explained by the accumulation of misincorporated complexes, rather than mistakes in mature RNA.


Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , Transcrição Genética , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Modelos Genéticos , RNA/genética , RNA/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Fatores de Elongação da Transcrição/metabolismo
15.
Transcription ; 8(2): 99-105, 2017 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-28072558

RESUMO

Pausing by RNA polymerase is a major mechanism that regulates transcription elongation but can cause conflicts with fellow RNA polymerases and other cellular machineries. Here, we summarize our recent finding that misincorporation could be a major source of transcription pausing in vivo, and discuss the role of misincorporation-induced pausing.


Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , Elongação da Transcrição Genética/fisiologia , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fatores de Elongação da Transcrição/genética , Fatores de Elongação da Transcrição/metabolismo
16.
Nucleic Acids Res ; 44(6): 2577-92, 2016 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-26609136

RESUMO

Coupled transcription and translation in bacteria are tightly regulated. Some small RNAs (sRNAs) control aspects of this coupling by modifying ribosome access or inducing degradation of the message. Here, we show that sRNA IsrA (IS61 or McaS) specifically associates with core enzyme of RNAP in vivo and in vitro, independently of σ factor and away from the main nucleic-acids-binding channel of RNAP. We also show that, in the cells, IsrA exists as ribonucleoprotein particles (sRNPs), which involve a defined set of proteins including Hfq, S1, CsrA, ProQ and PNPase. Our findings suggest that IsrA might be directly involved in transcription or can participate in regulation of gene expression by delivering proteins associated with it to target mRNAs through its interactions with transcribing RNAP and through regions of sequence-complementarity with the target. In this eukaryotic-like model only in the context of a complex with its target, IsrA and its associated proteins become active. In this manner, in the form of sRNPs, bacterial sRNAs could regulate a number of targets with various outcomes, depending on the set of associated proteins.


Assuntos
RNA Polimerases Dirigidas por DNA/genética , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , RNA Bacteriano/genética , Pequeno RNA não Traduzido/genética , Ribonucleoproteínas/genética , Sequência de Bases , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Fator Proteico 1 do Hospedeiro/genética , Fator Proteico 1 do Hospedeiro/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Dados de Sequência Molecular , Polirribonucleotídeo Nucleotidiltransferase/genética , Polirribonucleotídeo Nucleotidiltransferase/metabolismo , Biossíntese de Proteínas , RNA Bacteriano/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Pequeno RNA não Traduzido/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Ribonucleoproteínas/metabolismo , Fator sigma/genética , Fator sigma/metabolismo , Transcrição Genética
17.
Methods ; 86: 51-9, 2015 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-26080048

RESUMO

The various properties of RNA polymerase (RNAP) complexes with nucleic acids during different stages of transcription involve various types of regulation and different cross-talk with other cellular entities and with fellow RNAP molecules. The interactions of transcriptional apparatus with the translational machinery have been focused mainly in terms of outcomes of gene expression, whereas the study of the physical interaction of the ribosome and the RNAP remains obscure partly due to the lack of a system that allows such observations. In this article we will describe the methodology needed to set up a pure, transcription-coupled-to-translation system in which the translocation of the ribosome can be performed in a step-wise manner towards RNAP allowing investigation of the interactions between the two machineries at colliding and non-colliding distances. In the same time RNAP can be put in various types of states, such as paused, roadblocked, backtracked, etc. The experimental system thus allows studying the effects of the ribosome on different aspects of transcription elongation and the effects by RNAP on translation.


Assuntos
RNA Polimerases Dirigidas por DNA/genética , Biossíntese de Proteínas , RNA Mensageiro/genética , Transcrição Genética , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli , Biologia Molecular/métodos , RNA Mensageiro/biossíntese , RNA Mensageiro/química , Ribossomos/genética , Ribossomos/metabolismo
18.
Biomolecules ; 5(3): 1195-209, 2015 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-26120903

RESUMO

Transcription elongation is regulated at several different levels, including control by various accessory transcription elongation factors. A distinct group of these factors interacts with the RNA polymerase secondary channel, an opening at the enzyme surface that leads to its active center. Despite investigation for several years, the activities and in vivo roles of some of these factors remain obscure. Here, we review the recent progress in understanding the functions of the secondary channel binding factors in bacteria. In particular, we highlight the surprising role of global regulator DksA in fidelity of RNA synthesis and the resolution of RNA polymerase traffic jams by the Gre factor. These findings indicate a potential link between transcription fidelity and collisions of the transcription and replication machineries.


Assuntos
RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/metabolismo , Fatores de Transcrição/metabolismo , Ligação Competitiva , Replicação do DNA , Ligação Proteica , Fatores de Transcrição/química , Transcrição Genética
19.
Nucleic Acids Res ; 43(13): 6299-308, 2015 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-26038312

RESUMO

Regulation of transcription elongation is based on response of RNA polymerase (RNAP) to various pause signals and is modulated by various accessory factors. Here we report that a 7 kDa protein p7 encoded by bacteriophage Xp10 acts as an elongation processivity factor of RNAP of host bacterium Xanthomonas oryzae, a major rice pathogen. Our data suggest that p7 stabilizes the upstream DNA duplex of the elongation complex thus disfavouring backtracking and promoting forward translocated states of the elongation complex. The p7-induced 'pushing' of RNAP and modification of RNAP contacts with the upstream edge of the transcription bubble lead to read-through of various types of pauses and termination signals and generally increase transcription processivity and elongation rate, contributing for transcription of an extremely long late genes operon of Xp10. Forward translocation was observed earlier upon the binding of unrelated bacterial elongation factor NusG, suggesting that this may be a general pathway of regulation of transcription elongation.


Assuntos
Bacteriófagos , RNA Polimerases Dirigidas por DNA/metabolismo , Elongação da Transcrição Genética , Proteínas Virais/metabolismo , DNA/metabolismo , Terminação da Transcrição Genética , Xanthomonas/enzimologia
20.
Methods Mol Biol ; 1276: 81-99, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25665559

RESUMO

RNA polymerase is a complex machinery, which is further embedded in interactions with other cellular components that interplay with either the transcribed DNA (DNA polymerases, topoisomerases, etc.) or the nascent RNA (RNA processing enzymes, ribosomes, etc.). In prokaryotes, coupling of transcription and translation is thought to play many regulatory roles but the mechanistic understanding of their interactions has been hindered by the lack of a defined experimental system. Here, we describe a pure transcription-coupled-to-translation system in which control of the ribosome has been achieved through its stepwise translocation towards RNA polymerase. This system can be used to study the effects of concurrent translation on RNA chain elongation and to elucidate the interface between the two macromolecular complexes.


Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , Técnicas In Vitro/métodos , Biologia Molecular/métodos , Biossíntese de Proteínas/fisiologia , Ribossomos/metabolismo , Transcrição Genética/fisiologia , Sequência de Bases , Dados de Sequência Molecular , Regiões Promotoras Genéticas/genética
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