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1.
Chemistry ; 25(53): 12303-12307, 2019 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-31373735

RESUMO

Triplex forming oligonucleotides are used as a tool for gene regulation and in DNA nanotechnology. By incorporating artificial nucleic acids, target affinity and biological stability superior to that of natural DNA may be obtained. This work demonstrates how a chimeric clamp consisting of acyclic (L)-threoninol nucleic acid (aTNA) and DNA can bind DNA and RNA by the formation of a highly stable triplex structure. The (L)-aTNA clamp is released from the target again by the addition of a releasing strand in a strand displacement type of reaction. It is shown that the clamp efficiently inhibits Bsu and T7 RNA polymerase activity and that polymerase activity is reactivated by displacing the clamp. The clamp was successfully applied to the regulation of luciferase expression by reversible binding to the mRNA. When targeting a sequence in the double stranded plasmid, 40 % downregulation of protein expression is achieved.


Assuntos
RNA Polimerases Dirigidas por DNA/química , DNA/química , Ácidos Nucleicos/química , RNA/química , Proteínas Virais/química , Amino Álcoois/química , Butileno Glicóis/química , RNA Polimerases Dirigidas por DNA/metabolismo , RNA Polimerases Dirigidas por DNA/farmacologia , Proteínas Virais/metabolismo , Proteínas Virais/farmacologia
2.
Nat Commun ; 10(1): 3001, 2019 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-31278272

RESUMO

Type III-A CRISPR-Cas systems are prokaryotic RNA-guided adaptive immune systems that use a protein-RNA complex, Csm, for transcription-dependent immunity against foreign DNA. Csm can cleave RNA and single-stranded DNA (ssDNA), but whether it targets one or both nucleic acids during transcription elongation is unknown. Here, we show that binding of a Thermus thermophilus (T. thermophilus) Csm (TthCsm) to a nascent transcript in a transcription elongation complex (TEC) promotes tethering but not direct contact of TthCsm with RNA polymerase (RNAP). Biochemical experiments show that both TthCsm and Staphylococcus epidermidis (S. epidermidis) Csm (SepCsm) cleave RNA transcripts, but not ssDNA, at the transcription bubble. Taken together, these results suggest that Type III systems primarily target transcripts, instead of unwound ssDNA in TECs, for immunity against double-stranded DNA (dsDNA) phages and plasmids. This reveals similarities between Csm and eukaryotic RNA interference, which also uses RNA-guided RNA targeting to silence actively transcribed genes.


Assuntos
Imunidade Adaptativa/genética , Sistemas CRISPR-Cas/genética , Staphylococcus epidermidis/genética , Thermus thermophilus/genética , Elongação da Transcrição Genética/imunologia , Bacteriófagos/imunologia , Sistemas CRISPR-Cas/imunologia , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/imunologia , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/imunologia , DNA de Cadeia Simples/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Plasmídeos/imunologia , RNA Guia/genética , RNA Guia/imunologia , RNA Guia/metabolismo , Staphylococcus epidermidis/imunologia , Thermus thermophilus/imunologia
3.
Nat Commun ; 10(1): 3048, 2019 07 11.
Artigo em Inglês | MEDLINE | ID: mdl-31296855

RESUMO

Bacteriophages typically hijack the host bacterial transcriptional machinery to regulate their own gene expression and that of the host bacteria. The structural basis for bacteriophage protein-mediated transcription regulation-in particular transcription antitermination-is largely unknown. Here we report the 3.4 Å and 4.0 Å cryo-EM structures of two bacterial transcription elongation complexes (P7-NusA-TEC and P7-TEC) comprising the bacteriophage protein P7, a master host-transcription regulator encoded by bacteriophage Xp10 of the rice pathogen Xanthomonas oryzae pv. Oryzae (Xoo) and discuss the mechanisms by which P7 modulates the host bacterial RNAP. The structures together with biochemical evidence demonstrate that P7 prevents transcription termination by plugging up the RNAP RNA-exit channel and impeding RNA-hairpin formation at the intrinsic terminator. Moreover, P7 inhibits transcription initiation by restraining RNAP-clamp motions. Our study reveals the structural basis for transcription antitermination by phage proteins and provides insights into bacterial transcription regulation.


Assuntos
Proteínas de Bactérias/metabolismo , Bacteriófagos/genética , Fatores de Elongação da Transcrição/metabolismo , Proteínas Virais/metabolismo , Xanthomonas/genética , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/ultraestrutura , Microscopia Crioeletrônica , RNA Polimerases Dirigidas por DNA/isolamento & purificação , RNA Polimerases Dirigidas por DNA/metabolismo , RNA Polimerases Dirigidas por DNA/ultraestrutura , Regulação Bacteriana da Expressão Gênica , Regulação Viral da Expressão Gênica , Interações entre Hospedeiro e Microrganismos/genética , Oryza/microbiologia , Estrutura Secundária de Proteína , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Regiões Terminadoras Genéticas/genética , Transcrição Genética , Fatores de Elongação da Transcrição/isolamento & purificação , Fatores de Elongação da Transcrição/ultraestrutura , Proteínas Virais/isolamento & purificação , Proteínas Virais/ultraestrutura , Xanthomonas/virologia
4.
Gene ; 713: 143951, 2019 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-31269464

RESUMO

Rifampicin (RIF) is still a first line of antibiotic in the treatment of bacterial diseases, in particular the Mycobacterial infections. The antimicrobial activity of RIF is attributed to its ability to inhibit transcription by binding to the ß subunit of bacterial RNA polymerase (encoded by rpoB). Continued use of this drug resulted in the emergence of RIF resistant rpoB mutations in a high frequency that compels the use of RIF almost exclusively in drug combinations. As of date, a broad array of rif mutations have been isolated and characterized by different research groups. Studies on rpoB mutations strengthen the view that the ß subunit of RNA polymerase (RNAP) is very crucial in modulating transcription thereby leading to differential gene expression. Very recently we have reported the transcriptome profile of rpoB12 mutant that provides molecular evidence that presence of rpoB12 mutation modulates the transcription of about 450 genes. Here we present a maiden report that rpoB mutations that substitute Tyr at the Rif binding pocket (RBP) of ß subunit of RNA polymerase are able to suppress the over-production of colanic acid capsular polysaccharide (Ces phenotype) in Δlon mutant of Escherichia coli. Further analyses of the rif mutants involving their growth pattern on LB at higher temperature (42 °C), LB media without NaCl, survival in LB media with acidic pH (pH - 3) and motility revealed that only rpoB12 (His526Tyr) and rpoB137 (Ser522Tyr) affected all the above mentioned physiological parameters in addition to the elicitation of Ces phenotype. These two rif mutations confer fast movement to RNAP and they bear Tyr as the substituted amino acid in the RBP. This is perhaps the first study that brings out the possible role of Tyr in the RBP and its participation in the global gene expression. This study also envisages the point that amino acid residues that share the properties of Tyr in the RBP can be employed as a tool to bring out differential gene expression which would certainly have basic and applied values for the mankind.


Assuntos
RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Proteínas de Escherichia coli/genética , Escherichia coli/metabolismo , Mutação , Rifampina/farmacologia , Tirosina/metabolismo , Antibióticos Antituberculose/farmacologia , Farmacorresistência Bacteriana , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Fenótipo , RNA Bacteriano , Tirosina/genética
5.
Nature ; 572(7767): 136-140, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31316204

RESUMO

Many genome-processing reactions, including transcription, replication and repair, generate DNA rotation. Methods that directly measure DNA rotation, such as rotor bead tracking1-3, angular optical trapping4 and magnetic tweezers5, have helped to unravel the action mechanisms of a range of genome-processing enzymes that includes RNA polymerase (RNAP)6, gyrase2, a viral DNA packaging motor7 and DNA recombination enzymes8. Despite the potential of rotation measurements to transform our understanding of genome-processing reactions, measuring DNA rotation remains a difficult task. The time resolution of existing methods is insufficient for tracking the rotation induced by many enzymes under physiological conditions, and the measurement throughput is typically low. Here we introduce origami-rotor-based imaging and tracking (ORBIT), a method that uses fluorescently labelled DNA origami rotors to track DNA rotation at the single-molecule level with a time resolution of milliseconds. We used ORBIT to track the DNA rotations that result from unwinding by the RecBCD complex, a helicase that is involved in DNA repair9, as well as from transcription by RNAP. We characterized a series of events that occur during RecBCD-induced DNA unwinding-including initiation, processive translocation, pausing and backtracking-and revealed an initiation mechanism that involves reversible ATP-independent DNA unwinding and engagement of the RecB motor. During transcription by RNAP, we directly observed rotational steps that correspond to the unwinding of single base pairs. We envisage that ORBIT will enable studies of a wide range of interactions between proteins and DNA.


Assuntos
DNA/análise , DNA/metabolismo , Exodesoxirribonuclease V/metabolismo , Genoma/genética , Conformação de Ácido Nucleico , Rotação , Pareamento de Bases , DNA/química , Quebras de DNA de Cadeia Dupla , DNA Helicases/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Transcrição Genética
6.
Nat Commun ; 10(1): 2925, 2019 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-31266960

RESUMO

Bacteriophage Q protein engages σ-dependent paused RNA polymerase (RNAP) by binding to a DNA site embedded in late gene promoter and renders RNAP resistant to termination signals. Here, we report a single-particle cryo-electron microscopy (cryo-EM) structure of an intact Q-engaged arrested complex. The structure reveals key interactions responsible for σ-dependent pause, Q engagement, and Q-mediated transcription antitermination. The structure shows that two Q protomers (QI and QII) bind to a direct-repeat DNA site and contact distinct elements of the RNA exit channel. Notably, QI forms a narrow ring inside the RNA exit channel and renders RNAP resistant to termination signals by prohibiting RNA hairpin formation in the RNA exit channel. Because the RNA exit channel is conserved among all multisubunit RNAPs, it is likely to serve as an important contact site for regulators that modify the elongation properties of RNAP in other organisms, as well.


Assuntos
Bacteriófagos/enzimologia , Códon de Terminação/genética , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/metabolismo , Transcrição Genética , Proteínas Virais/química , Proteínas Virais/metabolismo , Bacteriófagos/química , Bacteriófagos/genética , Códon de Terminação/metabolismo , Microscopia Crioeletrônica , RNA Polimerases Dirigidas por DNA/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Escherichia coli/virologia , Regiões Promotoras Genéticas , Proteínas Virais/genética
7.
Biochemistry (Mosc) ; 84(4): 426-434, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31228934

RESUMO

The bacterium Escherichia coli has seven σ subunits that bind core RNA polymerase and are necessary for promoter recognition. It was previously shown that the σ70 and σ38 subunits can also interact with the transcription elongation complex (TEC) and stimulate pausing by recognizing DNA sequences similar to the -10 element of promoters. In this study, we analyzed the ability of the σ32, σ28, and σ24 subunits to induce pauses in reconstituted TECs containing corresponding -10 consensus elements. It was found that the σ24 subunit can induce a transcriptional pause depending on the presence of the -10 element. Pause formation is suppressed by the Gre factors, suggesting that the paused complex adopts a backtracked conformation. Some natural promoters contain potential signals of σ24-dependent pauses in the initially transcribed regions, suggesting that such pauses may have regulatory functions in transcription.


Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Transcrição Genética/fisiologia , Sequência de Bases , DNA/metabolismo , RNA Polimerases Dirigidas por DNA/genética , Proteínas de Escherichia coli/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Fator sigma/genética , Fator sigma/metabolismo , Fatores de Transcrição/metabolismo
8.
Nat Commun ; 10(1): 2630, 2019 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-31201314

RESUMO

Phytochromes initiate chloroplast biogenesis by activating genes encoding the photosynthetic apparatus, including photosynthesis-associated plastid-encoded genes (PhAPGs). PhAPGs are transcribed by a bacterial-type RNA polymerase (PEP), but how phytochromes in the nucleus activate chloroplast gene expression remains enigmatic. We report here a forward genetic screen in Arabidopsis that identified NUCLEAR CONTROL OF PEP ACTIVITY (NCP) as a necessary component of phytochrome signaling for PhAPG activation. NCP is dual-targeted to plastids and the nucleus. While nuclear NCP mediates the degradation of two repressors of chloroplast biogenesis, PIF1 and PIF3, NCP in plastids promotes the assembly of the PEP complex for PhAPG transcription. NCP and its paralog RCB are non-catalytic thioredoxin-like proteins that diverged in seed plants to adopt nonredundant functions in phytochrome signaling. These results support a model in which phytochromes control PhAPG expression through light-dependent double nuclear and plastidial switches that are linked by evolutionarily conserved and dual-localized regulatory proteins.


Assuntos
Proteínas de Arabidopsis/metabolismo , Cloroplastos/metabolismo , Chaperonas Moleculares/metabolismo , Fitocromo/metabolismo , Transcrição Genética/fisiologia , Arabidopsis/fisiologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , Cloroplastos/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Luz , Fotossíntese/fisiologia , Plantas Geneticamente Modificadas , Plastídeos/genética , Plastídeos/metabolismo , Transdução de Sinais/fisiologia , Transcrição Genética/efeitos da radiação
9.
Nat Commun ; 10(1): 2629, 2019 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-31201355

RESUMO

Light initiates chloroplast biogenesis by activating photosynthesis-associated genes encoded by not only the nuclear but also the plastidial genome, but how photoreceptors control plastidial gene expression remains enigmatic. Here we show that the photoactivation of phytochromes triggers the expression of photosynthesis-associated plastid-encoded genes (PhAPGs) by stimulating the assembly of the bacterial-type plastidial RNA polymerase (PEP) into a 1000-kDa complex. Using forward genetic approaches, we identified REGULATOR OF CHLOROPLAST BIOGENESIS (RCB) as a dual-targeted nuclear/plastidial phytochrome signaling component required for PEP assembly. Surprisingly, RCB controls PhAPG expression primarily from the nucleus by interacting with phytochromes and promoting their localization to photobodies for the degradation of the transcriptional regulators PIF1 and PIF3. RCB-dependent PIF degradation in the nucleus signals the plastids for PEP assembly and PhAPG expression. Thus, our findings reveal the framework of a nucleus-to-plastid anterograde signaling pathway by which phytochrome signaling in the nucleus controls plastidial transcription.


Assuntos
Proteínas de Arabidopsis/metabolismo , Cloroplastos/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Fitocromo/metabolismo , Tiorredoxinas/metabolismo , Transcrição Genética/fisiologia , Arabidopsis/fisiologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Núcleo Celular/metabolismo , Cloroplastos/genética , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Luz , Fotossíntese/fisiologia , Plantas Geneticamente Modificadas , Plastídeos/genética , Plastídeos/metabolismo , Proteólise , Transdução de Sinais/fisiologia , Transcrição Genética/efeitos da radiação
10.
Biochimie ; 163: 137-141, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31181235

RESUMO

RNA amplification has extensive applications on biochemistry and its related fields. Here, we present an isothermal strategy named rolling circle reverse transcription-mediated RNA amplification (RCRT-MRA) to amplify small RNAs with accurate length and sequence. The target RNA and complementary DNA were circularized to serve as amplicons replicated via the rolling circle mechanism. The transcription product consisting of tandemly repeated RNA units, was monomerized by site-specific cleavage to generate amplified RNA with authentic length and sequence. T4 DNA ligase was chosen to circularize RNA template for its high efficiency and low cost. SuperScript IV reverse transcriptase was found to be able to catalyze the RCRT reaction on the circular RNA template, and the reaction efficiency was enhanced by adding the nicking enzyme, Nb.BbvCI to the RCRT system. E. Coli RNA polymerase, instead of the commonly used T7 RNA polymerase, was applied to synthesize long-strand RNA product for its high universality and processivity. Under the optimized conditions, small RNAs can be precisely amplified by 105∼6 folds. The fidelity of the established method was demonstrated by the accordance of the sequencing result and the initial RNA sequences. Free from expensive thermal cycler (necessary for RT-PCR-based amplification), precise replication of the initial RNA and high fidelity will enable the established strategy to be applied in RNA-seq, mRNA profiling, microarray analysis and RNA-based SELEX as well.


Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , Técnicas de Amplificação de Ácido Nucleico/métodos , RNA/metabolismo , Transcrição Reversa , DNA Circular/metabolismo , Escherichia coli/enzimologia , Proteínas de Escherichia coli/metabolismo
11.
Plant Physiol Biochem ; 141: 325-331, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31207493

RESUMO

Type 2C protein phosphatases (PP2Cs) counteract protein kinases, thereby inhibiting the abscisic acid (ABA)-mediated response to abiotic stress in Arabidopsis thaliana. In the absence of stress, the promoters of PP2C genes (e.g., ABI1, ABI2, and HAI1) are negatively regulated by repressors that suppress gene transcription in a signal-independent manner. Quantitative reverse transcription PCR (RT-qPCR) and chromatin immunoprecipitation (ChIP) assays revealed that the levels of PP2C gene transcripts and RNA polymerase II (RNAPII) that stalled at the transcription start sites (TSS) of PP2C gene loci were increased under salt stress. The salt-induced increases in RNA polymerase-mediated transcription were reduced in 35S:AtMYB44 plants, confirming that AtMYB44 acts as a repressor of PP2C gene transcription. ChIP assays revealed that AtMYB44 repressors are released and nucleosomes are evicted from the promoter regions in response to salt stress. Under these conditions, histone H3 acetylation (H3ac) and methylation (H3K4me3) around the TSS regions significantly increased. The salt-induced increases in PP2C gene transcription were reduced in abf3 plants, indicating that ABF3 activates PP2C gene transcription. Overall, our data indicate that salt stress converts PP2C gene chromatin from a repressor-associated suppression status to an activator-mediated transcription status. In addition, we observed that the Arabidopsis mutant brm-3, which is moderately defective in SWI2/SNF2 chromatin remodeling ATPase BRAHMA (BRM) activity, produced more PP2C gene transcripts under salt stress conditions, indicating that BRM ATPase contributes to the repression of PP2C gene transcription.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Cromatina/química , Nucleossomos/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Estresse Salino , Trifosfato de Adenosina/química , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Montagem e Desmontagem da Cromatina , Metilação de DNA , RNA Polimerases Dirigidas por DNA/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Plantas Geneticamente Modificadas/genética , Regiões Promotoras Genéticas , Estresse Fisiológico , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Genética
12.
RNA ; 25(9): 1192-1201, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31239298

RESUMO

Protein kinase RNA-activated (PKR) is an interferon-inducible kinase that is potently activated by long double-stranded RNA (dsRNA). In a previous study, we found that snoRNAs exhibit increased association with PKR in response to metabolic stress. While it was unclear if snoRNAs also activated PKR in cells, activation in vitro was observed. snoRNAs do not exhibit the double-stranded character typically required for activation of PKR, but some studies suggest such RNAs can activate PKR if triphosphorylated at the 5' terminus, or if they are able to form intermolecular dimers. To interrogate the mechanism of PKR activation by snoRNAs in vitro we focused on SNORD113. Using multiple methods for defining the 5'-phosphorylation state, we find that activation of PKR by SNORD113 does not require a 5'-triphosphate. Gel purification from a native gel followed by analysis using analytical ultracentrifugation showed that dimerization was also not responsible for activation. We isolated distinct conformers of SNORD113 from a native polyacrylamide gel and tracked the activating species to dsRNA formed from antisense RNA synthesized during in vitro transcription with T7 RNA polymerase. Similar studies with additional snoRNAs and small RNAs showed the generality of our results. Our studies suggest that a 5' triphosphate is not an activating ligand for PKR, and emphasize the insidious nature of antisense contamination.


Assuntos
Ativação Enzimática/genética , Polifosfatos/metabolismo , eIF-2 Quinase/genética , eIF-2 Quinase/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Dimerização , Humanos , Ligantes , Fosforilação/genética , Ligação Proteica/genética , RNA de Cadeia Dupla/genética , RNA Nucleolar Pequeno/genética , Transcrição Genética/genética , Ultracentrifugação/métodos , Proteínas Virais/metabolismo
13.
Phys Rev E ; 99(5-1): 052122, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31212543

RESUMO

We develop a theoretical framework, based on an exclusion process, that is motivated by a biological phenomenon called transcript slippage (TS). In this model a discrete lattice represents a DNA strand while each of the particles that hop on it unidirectionally, from site to site, represents a RNA polymerase (RNAP). While walking like a molecular motor along a DNA track in a step-by-step manner, a RNAP simultaneously synthesizes an RNA chain; in each forward step it elongates the nascent RNA molecule by one unit, using the DNA track also as the template. At some special "slippery" position on the DNA, which we represent as a defect on the lattice, a RNAP can lose its grip on the nascent RNA and the latter's consequent slippage results in a final product that is either longer or shorter than the corresponding DNA template. We develop an exclusion model for RNAP traffic where the kinetics of the system at the defect site captures key features of TS events. We demonstrate the interplay of the crowding of RNAPs and TS. A RNAP has to wait at the defect site for a longer period in more congested RNAP traffic, thereby increasing the likelihood of its suffering a larger number of TS events. The qualitative trends of some of our results for a simple special case of our model are consistent with experimental observations. The general theoretical framework presented here will be useful for guiding future experimental queries and for analysis of the experimental data with more detailed versions of the same model.


Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , Modelos Genéticos , DNA/genética , DNA/metabolismo , Cinética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
14.
Zhejiang Da Xue Xue Bao Yi Xue Ban ; 48(1): 44-49, 2019 May 25.
Artigo em Chinês | MEDLINE | ID: mdl-31102357

RESUMO

Rifamycins, a group of bacterial RNA polymerase inhibitors, are the firstline antimicrobial drugs to treat tuberculosis. In light of the emergence of rifamycinresistant bacteria, development of new RNA polymerase inhibitors that kill rifamycinresistant bacteria with high bioavailability is urgent. Structural analysis of bacterial RNA polymerase in complex with inhibitors by crystallography and cryo-EM indicates that RNA polymerase inhibitors function through five distinct molecular mechanisms:inhibition of the extension of short RNA; competition with substrates; inhibition of the conformational change of the'bridge helix'; inhibition of clamp opening;inhibition of clamp closure. This article reviews the research progress of these five groups of RNA polymerase inhibitors to provide references for the modification of existing RNA polymerase inhibitors and the discovery of new RNA polymerase inhibitors.


Assuntos
RNA Polimerases Dirigidas por DNA , Descoberta de Drogas , RNA Bacteriano , Antituberculosos/uso terapêutico , Bactérias/efeitos dos fármacos , Bactérias/enzimologia , RNA Polimerases Dirigidas por DNA/metabolismo , Descoberta de Drogas/tendências , Farmacorresistência Bacteriana , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Humanos , Tuberculose/tratamento farmacológico , Tuberculose/enzimologia
15.
Int J Mol Sci ; 20(10)2019 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-31137816

RESUMO

The coupling of transcription and translation is more than mere translation of an mRNA that is still being transcribed. The discovery of physical interactions between RNA polymerase and ribosomes has spurred renewed interest into this long-standing paradigm of bacterial molecular biology. Here, we provide a concise presentation of recent insights gained from super-resolution microscopy, biochemical, and structural work, including cryo-EM studies. Based on the presented data, we put forward a dynamic model for the interaction between RNA polymerase and ribosomes, in which the interactions are repeatedly formed and broken. Furthermore, we propose that long intervening nascent RNA will loop out and away during the forming the interactions between the RNA polymerase and ribosomes. By comparing the effect of the direct interactions between RNA polymerase and ribosomes with those that transcription factors NusG and RfaH mediate, we submit that two distinct modes of coupling exist: Factor-free and factor-mediated coupling. Finally, we provide a possible framework for transcription-translation coupling and elude to some open questions in the field.


Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Fatores de Alongamento de Peptídeos/metabolismo , Ribossomos/metabolismo , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Biossíntese de Proteínas , Transcrição Genética
16.
Genes Dev ; 33(11-12): 684-704, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-31048545

RESUMO

DNA double-strand breaks (DSBs) at RNA polymerase II (RNAPII) transcribed genes lead to inhibition of transcription. The DNA-dependent protein kinase (DNA-PK) complex plays a pivotal role in transcription inhibition at DSBs by stimulating proteasome-dependent eviction of RNAPII at these lesions. How DNA-PK triggers RNAPII eviction to inhibit transcription at DSBs remains unclear. Here we show that the HECT E3 ubiquitin ligase WWP2 associates with components of the DNA-PK and RNAPII complexes and is recruited to DSBs at RNAPII transcribed genes. In response to DSBs, WWP2 targets the RNAPII subunit RPB1 for K48-linked ubiquitylation, thereby driving DNA-PK- and proteasome-dependent eviction of RNAPII. The lack of WWP2 or expression of nonubiquitylatable RPB1 abrogates the binding of nonhomologous end joining (NHEJ) factors, including DNA-PK and XRCC4/DNA ligase IV, and impairs DSB repair. These findings suggest that WWP2 operates in a DNA-PK-dependent shutoff circuitry for RNAPII clearance that promotes DSB repair by protecting the NHEJ machinery from collision with the transcription machinery.


Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Proteína Quinase Ativada por DNA/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Proteínas Nucleares/metabolismo , RNA Polimerase II/metabolismo , Transcrição Genética , Ubiquitina-Proteína Ligases/metabolismo , Linhagem Celular Transformada , Linhagem Celular Tumoral , Humanos , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitinação
17.
Eur J Med Chem ; 171: 401-419, 2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-30928711

RESUMO

GSK3082 - a hepatitis C virus RNA polymerase inhibitor - and a series of analogues with structural diversity at the 5-position were prepared from a 2,2,4,5-tetrasubstituted pyrrolidine obtained with a well-defined stereochemistry from the 1,3-dipolar cycloaddition of the chiral imino ester derived from leucine tert-butyl ester and (R)-2,3-O-isopropylideneglyceraldehyde with methyl acrylate. The chiral 2,2-dimethyl-1,3-dioxolane moiety provided by the glyceraldehyde served as a synthetic equivalent for different substituents and functional groups and these transformations usually required mild reaction conditions and simple work-up procedures. The inhibitory activity of the resulting GSK3082 analogues was studied in vitro in a cell-based assay of the subgenomic HCV RNA replication system. Some of the analogues showed good inhibitory activity with IC50 values in the nanomolar concentration range.


Assuntos
Antivirais/farmacologia , RNA Polimerases Dirigidas por DNA/antagonistas & inibidores , Hepacivirus/efeitos dos fármacos , Antivirais/síntese química , Antivirais/química , RNA Polimerases Dirigidas por DNA/metabolismo , Relação Dose-Resposta a Droga , Hepacivirus/enzimologia , Testes de Sensibilidade Microbiana , Estrutura Molecular , Estereoisomerismo , Relação Estrutura-Atividade , Replicação Viral/efeitos dos fármacos
18.
Nat Commun ; 10(1): 1133, 2019 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-30850604

RESUMO

Genome editing for therapeutic applications often requires cleavage within a narrow sequence window. Here, to enable such high-precision targeting with zinc-finger nucleases (ZFNs), we have developed an expanded set of architectures that collectively increase the configurational options available for design by a factor of 64. These new architectures feature the functional attachment of the FokI cleavage domain to the amino terminus of one or both zinc-finger proteins (ZFPs) in the ZFN dimer, as well as the option to skip bases between the target triplets of otherwise adjacent fingers in each zinc-finger array. Using our new architectures, we demonstrate targeting of an arbitrarily chosen 28 bp genomic locus at a density that approaches 1.0 (i.e., efficient ZFNs available for targeting almost every base step). We show that these new architectures may be used for targeting three loci of therapeutic significance with a high degree of precision, efficiency, and specificity.


Assuntos
Desoxirribonucleases de Sítio Específico do Tipo II/genética , Edição de Genes/métodos , Genoma Humano , Engenharia de Proteínas/métodos , Nucleases de Dedos de Zinco/genética , Pareamento de Bases , Sequência de Bases , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Loci Gênicos , Biblioteca Genômica , Humanos , Mutação INDEL , Células K562 , Biblioteca de Peptídeos , Plasmídeos/química , Plasmídeos/metabolismo , Transformação Genética , Proteínas Virais/genética , Proteínas Virais/metabolismo , Nucleases de Dedos de Zinco/metabolismo
19.
Nat Commun ; 10(1): 1153, 2019 03 11.
Artigo em Inglês | MEDLINE | ID: mdl-30858373

RESUMO

Bacterial RNA polymerase employs extra-cytoplasmic function (ECF) σ factors to regulate context-specific gene expression programs. Despite being the most abundant and divergent σ factor class, the structural basis of ECF σ factor-mediated transcription initiation remains unknown. Here, we determine a crystal structure of Mycobacterium tuberculosis (Mtb) RNAP holoenzyme comprising an RNAP core enzyme and the ECF σ factor σH (σH-RNAP) at 2.7 Å, and solve another crystal structure of a transcription initiation complex of Mtb σH-RNAP (σH-RPo) comprising promoter DNA and an RNA primer at 2.8 Å. The two structures together reveal the interactions between σH and RNAP that are essential for σH-RNAP holoenzyme assembly as well as the interactions between σH-RNAP and promoter DNA responsible for stringent promoter recognition and for promoter unwinding. Our study establishes that ECF σ factors and primary σ factors employ distinct mechanisms for promoter recognition and for promoter unwinding.


Assuntos
Proteínas de Bactérias/química , RNA Polimerases Dirigidas por DNA/química , Regulação Bacteriana da Expressão Gênica , Mycobacterium tuberculosis/genética , Fator sigma/química , Iniciação da Transcrição Genética , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X , DNA Bacteriano/química , DNA Bacteriano/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Holoenzimas/química , Holoenzimas/metabolismo , Modelos Moleculares , Regiões Promotoras Genéticas/genética , Fator sigma/metabolismo
20.
Nat Commun ; 10(1): 1207, 2019 03 14.
Artigo em Inglês | MEDLINE | ID: mdl-30872584

RESUMO

In bacteria, transcription-coupled repair of DNA lesions initiates after the Mfd protein removes RNA polymerases (RNAPs) stalled at the lesions. The bacterial RNA helicase, Rho, is a transcription termination protein that dislodges the elongation complexes. Here, we show that Rho dislodges the stalled RNAPs at DNA lesions. Strains defective in both Rho and Mfd are susceptible to DNA-damaging agents and are inefficient in repairing or propagating UV-damaged DNA. In vitro transcription assays show that Rho dissociates the stalled elongation complexes at the DNA lesions. We conclude that Rho-dependent termination recycles stalled RNAPs, which might facilitate DNA repair and other DNA-dependent processes essential for bacterial cell survival. We surmise that Rho might compete with, or augment, the Mfd function.


Assuntos
Reparo do DNA/fisiologia , RNA Polimerases Dirigidas por DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/fisiologia , Terminação da Transcrição Genética/fisiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/efeitos da radiação , DNA Bacteriano/metabolismo , Proteínas de Escherichia coli/genética , Mitomicina/farmacologia , Mutação , Fatores de Alongamento de Peptídeos/genética , Fatores de Alongamento de Peptídeos/metabolismo , RNA Bacteriano/biossíntese , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Raios Ultravioleta/efeitos adversos
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