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1.
Bioorg Med Chem Lett ; 30(7): 126986, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32046903

RESUMO

Our HCV research program investigated novel 2'-dihalogenated nucleoside HCV polymerase inhibitors and identified compound 1, a 5'-phosphoramidate prodrug of 2'-deoxy-2'-α-bromo-ß-chloro uridine. Although 1 had a favorable in vitro activity profile in HCV replicons, oral dosing in dog resulted in low levels of the active 5'-triphosphate (TP) in liver. Metabolism studies using human hepatocytes provided a simple assay for screening alternative phosphoramidate prodrug analogs. Compounds that produced high TP concentrations in hepatocytes were tested in dog liver biopsy studies. This method identified 2-aminoisobutyric acid ethyl ester (AIBEE) phosphoramidate prodrug 14, which provided 100-fold higher TP concentrations in dog liver in comparison to 1 (4 and 24 h after 5 mg/kg oral dose).


Assuntos
Antivirais/farmacologia , Desoxiuridina/análogos & derivados , Desoxiuridina/farmacologia , Inibidores Enzimáticos/farmacologia , Hepacivirus/efeitos dos fármacos , Pró-Fármacos/farmacologia , Ácidos Aminoisobutíricos/metabolismo , Ácidos Aminoisobutíricos/farmacocinética , Ácidos Aminoisobutíricos/farmacologia , Animais , Antivirais/metabolismo , Antivirais/farmacocinética , Desoxiuridina/metabolismo , Desoxiuridina/farmacocinética , Cães , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacocinética , Hepacivirus/enzimologia , Hepatócitos/metabolismo , Humanos , Fígado/metabolismo , Testes de Sensibilidade Microbiana , Compostos Organofosforados/metabolismo , Compostos Organofosforados/farmacocinética , Compostos Organofosforados/farmacologia , Pró-Fármacos/metabolismo , Pró-Fármacos/farmacocinética , RNA Polimerase Dependente de RNA/antagonistas & inibidores , Proteínas não Estruturais Virais/antagonistas & inibidores , Replicação Viral/efeitos dos fármacos
2.
Bioorg Med Chem ; 28(1): 115208, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31740203

RESUMO

Hepatitis C virus (HCV) nucleoside inhibitors have been a key focus of nearly 2 decades of HCV drug research due to a high barrier to drug resistance and pan-genotypic activity profile provided by molecules in this drug class. Our investigations focused on several potent 2'-halogenated uridine-based HCV polymerase inhibitors, resulting in the discovery of novel 2'-deoxy-2'-dihalo-uridine analogs that are potent inhibitors in replicon assays for all genotypes. Further studies to improve in vivo performance of these nucleoside inhibitors identified aminoisobutyric acid ethyl ester (AIBEE) phosphoramidate prodrugs 18a and 18c, which provide high levels of the active triphosphate in dog liver. AIBEE prodrug 18c was compared with sofosbuvir (1) by co-dosing both compounds by oral administration in dog (5 mg/kg each) and measuring liver concentrations of the active triphosphate metabolite at both 4 and 24 h post dosing. In this study, 18c provided liver triphosphate concentrations that were 6-fold higher than sofosbuvir (1) at both biopsy time points, suggesting that 18c could be a highly effective agent for treating HCV infected patients in the clinic.


Assuntos
Antivirais/farmacologia , Hepacivirus/efeitos dos fármacos , Pró-Fármacos/farmacologia , Uridina/farmacologia , Antivirais/síntese química , Antivirais/química , Relação Dose-Resposta a Droga , Hepatócitos/efeitos dos fármacos , Humanos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Pró-Fármacos/síntese química , Pró-Fármacos/química , Relação Estrutura-Atividade , Uridina/análogos & derivados , Uridina/química , Replicação Viral/efeitos dos fármacos
3.
Mol Cell ; 37(1): 21-33, 2010 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-20129052

RESUMO

Cold induction of cspA, the paradigm Escherichia coli cold-shock gene, is mainly subject to posttranscriptional control, partly promoted by cis-acting elements of its transcript, whose secondary structure at 37 degrees C and at cold-shock temperature has been elucidated here by enzymatic and chemical probing. The structures, which were also validated by mutagenesis, demonstrate that cspA mRNA undergoes a temperature-dependent structural rearrangement, likely resulting from stabilization in the cold of an otherwise thermodynamically unstable folding intermediate. At low temperature, the "cold-shock" structure is more efficiently translated and somewhat less susceptible to degradation than the 37 degrees C structure. Overall, our data shed light on a molecular mechanism at the basis of the cold-shock response, indicating that cspA mRNA is able to sense temperature downshifts, adopting functionally distinct structures at different temperatures, even without the aid of trans-acting factors. Unlike with other previously studied RNA thermometers, these structural rearrangements do not result from melting of hairpin structures.


Assuntos
Temperatura Baixa , Proteínas de Escherichia coli/fisiologia , Escherichia coli/genética , Proteínas de Choque Térmico/fisiologia , Biossíntese de Proteínas , RNA Mensageiro/fisiologia , Regiões 5' não Traduzidas , Aclimatação , Proteínas e Peptídeos de Choque Frio , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Proteínas de Choque Térmico/genética , Modelos Genéticos , Conformação de Ácido Nucleico , RNA Mensageiro/química
4.
Artigo em Inglês | MEDLINE | ID: mdl-28193664

RESUMO

Pibrentasvir (ABT-530) is a novel and pan-genotypic hepatitis C virus (HCV) NS5A inhibitor with 50% effective concentration (EC50) values ranging from 1.4 to 5.0 pM against HCV replicons containing NS5A from genotypes 1 to 6. Pibrentasvir demonstrated similar activity against a panel of chimeric replicons containing HCV NS5A of genotypes 1 to 6 from clinical samples. Resistance selection studies were conducted using HCV replicon cells with NS5A from genotype 1a, 1b, 2a, 2b, 3a, 4a, 5a, or 6a at a concentration of pibrentasvir that was 10- or 100-fold over its EC50 for the respective replicon. With pibrentasvir at 10-fold over the respective EC50, only a small number of colonies (0.00015 to 0.0065% of input cells) with resistance-associated amino acid substitutions were selected in replicons containing genotype 1a, 2a, or 3a NS5A, and no viable colonies were selected in replicons containing NS5A from other genotypes. With pibrentasvir at 100-fold over the respective EC50, very few colonies (0.0002% of input cells) were selected by pibrentasvir in genotype 1a replicon cells while no colonies were selected in other replicons. Pibrentasvir is active against common resistance-conferring substitutions in HCV genotypes 1 to 6 that were identified for other NS5A inhibitors, including those at key amino acid positions 28, 30, 31, or 93. The combination of pibrentasvir with HCV inhibitors of other classes produced synergistic inhibition of HCV replication. In summary, pibrentasvir is a next-generation HCV NS5A inhibitor with potent and pan-genotypic activity, and it maintains activity against common amino acid substitutions of HCV genotypes 1 to 6 that are known to confer resistance to currently approved NS5A inhibitors.


Assuntos
Antivirais/farmacologia , Benzimidazóis/efeitos adversos , Benzimidazóis/farmacologia , Hepacivirus/efeitos dos fármacos , Pirrolidinas/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Substituição de Aminoácidos , Linhagem Celular Tumoral , Farmacorresistência Viral , Células Hep G2 , Hepacivirus/classificação , Hepacivirus/genética , Hepatite C/tratamento farmacológico , Humanos , Pirrolidinas/efeitos adversos , Proteínas não Estruturais Virais/genética
5.
Bioorg Med Chem Lett ; 26(22): 5462-5467, 2016 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-27780635

RESUMO

Research toward a next-generation HCV NS5A inhibitor has identified fluorobenzimidazole analogs that demonstrate potent, broad-genotype in vitro activity against HCV genotypes 1-6 replicons as well as HCV NS5A variants that are orders of magnitude less susceptible to inhibition by first-generation NS5A inhibitors in comparison to wild-type replicons. The fluorobenzimidazole inhibitors have improved pharmacokinetic properties in comparison to non-fluorinated benzimidazole analogs. Discovery of these inhibitors was facilitated by exploring SAR in a structurally simplified inhibitor series.


Assuntos
Antivirais/química , Antivirais/farmacologia , Benzimidazóis/química , Benzimidazóis/farmacologia , Hepacivirus/efeitos dos fármacos , Proteínas não Estruturais Virais/antagonistas & inibidores , Animais , Antivirais/farmacocinética , Benzimidazóis/farmacocinética , Cães , Genótipo , Halogenação , Hepacivirus/genética , Hepacivirus/metabolismo , Hepatite C/tratamento farmacológico , Humanos , Camundongos , Ratos , Replicon/efeitos dos fármacos , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo
6.
Nucleic Acids Res ; 42(12): 7884-93, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24920831

RESUMO

The adaptation against foreign nucleic acids by the CRISPR-Cas system (Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated proteins) depends on the insertion of foreign nucleic acid-derived sequences into the CRISPR array as novel spacers by still unknown mechanism. We identified and characterized in Escherichia coli intermediate states of spacer integration and mapped the integration site at the chromosomal CRISPR array in vivo. The results show that the insertion of new spacers occurs by site-specific nicking at both strands of the leader proximal repeat in a staggered way and is accompanied by joining of the resulting 5'-ends of the repeat strands with the 3'-ends of the incoming spacer. This concerted cleavage-ligation reaction depends on the metal-binding center of Cas1 protein and requires the presence of Cas2. By acquisition assays using plasmid-located CRISPR array with mutated repeat sequences, we demonstrate that the primary sequence of the first repeat is crucial for cleavage of the CRISPR array and the ligation of new spacer DNA.


Assuntos
Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Desoxirribonucleases/metabolismo , DNA/química , Escherichia coli/genética
7.
Mol Microbiol ; 91(4): 641-8, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24308327

RESUMO

6S RNA is a non-coding RNA, found in almost all phylogenetic branches of bacteria. Through its conserved secondary structure, resembling open DNA promoters, it binds to RNA polymerase and interferes with transcription at many promoters. That way, it functions as transcriptional regulator facilitating adaptation to stationary phase conditions. Strikingly, 6S RNA acts as template for the synthesis of small RNAs (pRNA), which trigger the disintegration of the inhibitory RNA polymerase-6S RNA complex releasing 6S RNA-dependent repression. The regulatory implications of 6S RNAs vary among different bacterial species depending on the lifestyle and specific growth conditions that they have to face. The influence of 6S RNA can be seen on many different processes including stationary growth, sporulation, light adaptation or intracellular growth of pathogenic bacteria. Recent structural and functional studies have yielded details of the interaction between E. coli 6S RNA and RNA polymerase. Genome-wide transcriptome analyses provided insight into the functional diversity of 6S RNAs. Moreover, the mechanism and physiological consequences of pRNA synthesis have been explored in several systems. A major function of 6S RNA as a guardian regulating the economic use of cellular resources under limiting conditions and stress emerges as a common perception from numerous recent studies.


Assuntos
Bactérias/genética , Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , RNA Bacteriano/metabolismo , Transcrição Gênica , RNA Polimerases Dirigidas por DNA/metabolismo , Ligação Proteica , RNA não Traduzido
8.
Antimicrob Agents Chemother ; 59(3): 1505-11, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25534735

RESUMO

Dasabuvir (ABT-333) is a nonnucleoside inhibitor of the RNA-dependent RNA polymerase encoded by the hepatitis C virus (HCV) NS5B gene. Dasabuvir inhibited recombinant NS5B polymerases derived from HCV genotype 1a and 1b clinical isolates, with 50% inhibitory concentration (IC50) values between 2.2 and 10.7 nM, and was at least 7,000-fold selective for the inhibition of HCV genotype 1 polymerases over human/mammalian polymerases. In the HCV subgenomic replicon system, dasabuvir inhibited genotype 1a (strain H77) and 1b (strain Con1) replicons with 50% effective concentration (EC50) values of 7.7 and 1.8 nM, respectively, with a 13-fold decrease in inhibitory activity in the presence of 40% human plasma. This level of activity was retained against a panel of chimeric subgenomic replicons that contained HCV NS5B genes from 22 genotype 1 clinical isolates from treatment-naive patients, with EC50s ranging between 0.15 and 8.57 nM. Maintenance of replicon-containing cells in medium containing dasabuvir at concentrations 10-fold or 100-fold greater than the EC50 resulted in selection of resistant replicon clones. Sequencing of the NS5B coding regions from these clones revealed the presence of variants, including C316Y, M414T, Y448C, Y448H, and S556G, that are consistent with binding to the palm I site of HCV polymerase. Consequently, dasabuvir retained full activity against replicons known to confer resistance to other polymerase inhibitors, including the S282T variant in the nucleoside binding site and the M423T, P495A, P495S, and V499A single variants in the thumb domain. The use of dasabuvir in combination with inhibitors targeting HCV NS3/NS4A protease (ABT-450 with ritonavir) and NS5A (ombitasvir) is in development for the treatment of HCV genotype 1 infections.


Assuntos
Antivirais/farmacologia , Hepacivirus/efeitos dos fármacos , Sulfonamidas/farmacologia , Uracila/análogos & derivados , Proteínas não Estruturais Virais/antagonistas & inibidores , 2-Naftilamina , Farmacorresistência Viral , Genótipo , Hepacivirus/classificação , Hepacivirus/genética , Humanos , Replicon/efeitos dos fármacos , Uracila/farmacologia
9.
Nucleic Acids Res ; 41(12): 6347-59, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23625968

RESUMO

The adaptive immunity of bacteria against foreign nucleic acids, mediated by CRISPR (clustered regularly interspaced short palindromic repeats), relies on the specific incorporation of short pieces of the invading foreign DNA into a special genomic locus, termed CRISPR array. The stored sequences (spacers) are subsequently used in the form of small RNAs (crRNAs) to interfere with the target nucleic acid. We explored the DNA-binding mechanism of the immunization protein Csn2 from the human pathogen Streptococcus agalactiae using different biochemical techniques, atomic force microscopic imaging and molecular dynamics simulations. The results demonstrate that the ring-shaped Csn2 tetramer binds DNA ends through its central hole and slides inward, likely by a screw motion along the helical path of the enclosed DNA. The presented data indicate an accessory function of Csn2 during integration of exogenous DNA by end-joining.


Assuntos
Proteínas de Bactérias/química , Proteínas de Ligação a DNA/química , DNA/química , Proteínas de Bactérias/metabolismo , Cálcio/metabolismo , DNA/metabolismo , DNA/ultraestrutura , Proteínas de Ligação a DNA/metabolismo , Microscopia de Força Atômica , Simulação de Dinâmica Molecular , Movimento (Física) , Ligação Proteica , Streptococcus agalactiae
10.
RNA Biol ; 11(5): 508-21, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24786589

RESUMO

Whereas, the majority of bacterial non-coding RNAs and functional RNA elements regulate post-transcriptional processes, either by interacting with other RNAs via base-pairing or through binding of small ligands (riboswitches), 6S RNAs affect transcription itself by binding to the housekeeping holoenzyme of RNA polymerase (RNAP). Remarkably, 6S RNAs serve as RNA templates for bacterial RNAP, giving rise to the de novo synthesis of short transcripts, termed pRNAs (product RNAs). Hence, 6S RNAs prompt the enzyme to act as an RNA-dependent RNA polymerase (RdRP). Synthesis of pRNAs exceeding a certain length limit (~13 nt) persistently rearrange the 6S RNA structure, which in turn, disrupts the 6S RNA:RNAP complex. This pRNA synthesis-mediated "reanimation" of sequestered RNAP molecules represents the conceivably fastest mechanism for resuming transcription in cells that enter a new exponential growth phase. The many different 6S RNAs found in a wide variety of bacteria do not share strong sequence homology but have in common a conserved rod-shaped structure with a large internal loop, termed the central bulge; this architecture mediates specific binding to the active site of RNAP. In this article, we summarize the overall state of knowledge as well as very recent findings on the structure, function, and physiological effects of 6S RNA examples from the two model organisms, Escherichia coli and Bacillus subtilis. Comparison of the presently known properties of 6S RNAs in the two organisms highlights common principles as well as diverse features.


Assuntos
Bacillus subtilis/genética , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , RNA Bacteriano/genética , RNA não Traduzido/genética , Transcrição Gênica , Bacillus subtilis/metabolismo , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/metabolismo , Conformação de Ácido Nucleico , Ligação Proteica , RNA Bacteriano/química , RNA Bacteriano/metabolismo , RNA não Traduzido/química , RNA não Traduzido/metabolismo , Moldes Genéticos
11.
Mol Microbiol ; 83(6): 1109-23, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22295907

RESUMO

The LysR-type transcription factor LeuO is involved in regulation of pathogenicity determinants and stress responses in Enterobacteriaceae, and acts as antagonist of the global repressor H-NS. Expression of the leuO gene is repressed by H-NS, and it is upregulated in stationary phase and under amino acid starvation conditions. Here, we show that the heterodimer of the FixJ/NarL-type transcription regulators RcsB and BglJ strongly activates expression of leuO and that RcsB-BglJ regulates additional loci. Activation of leuO by RcsB-BglJ is independent of the Rcs phosphorelay system. RcsB-BglJ binds to the leuO promoter region and activates one of two leuO promoters mapped in vivo. Moreover, LeuO antagonizes activation of leuO by RcsB-BglJ and acts as negative autoregulator in vivo and in vitro. Further, the H-NS paralogue StpA causes repression of leuO in addition to H-NS. Together, our data suggest a complex arrangement of regulatory elements and they indicate a feedback control mechanism of leuO expression.


Assuntos
Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas de Fímbrias/metabolismo , Regulação Bacteriana da Expressão Gênica , Transativadores/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fatores de Virulência/metabolismo , Sequência de Bases , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/antagonistas & inibidores , Proteínas de Fímbrias/antagonistas & inibidores , Proteínas de Fímbrias/genética , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Dados de Sequência Molecular , Óperon , Regiões Promotoras Genéticas , Ligação Proteica , Transativadores/genética , Ativação Transcricional , Fatores de Virulência/genética
12.
Bioorg Med Chem Lett ; 23(15): 4367-9, 2013 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-23791079

RESUMO

Described herein is the development of a potent non-nucleoside, small molecule inhibitor of genotype 1 HCV NS5B Polymerase. A 23 µM inhibitor that was active against HCV polymerase was further elaborated into a potent single-digit nanomolar inhibitor of HCV NS5B polymerase by additional manipulation of the R and R1 substituents. Subsequent modifications to improve physical properties were made in an attempt to achieve an acceptable pharmacokinetic profile.


Assuntos
Antivirais/síntese química , Hepacivirus/enzimologia , Uracila/análogos & derivados , Proteínas não Estruturais Virais/antagonistas & inibidores , Animais , Antivirais/química , Antivirais/farmacocinética , Meia-Vida , Hepacivirus/fisiologia , Ratos , Relação Estrutura-Atividade , Uracila/síntese química , Uracila/farmacocinética , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/efeitos dos fármacos
13.
Bioorg Med Chem Lett ; 23(12): 3487-90, 2013 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-23664214
14.
RNA Biol ; 10(5): 708-15, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23392250

RESUMO

Prokaryotic immunity against foreign nucleic acids mediated by clustered, regularly interspaced, short palindromic repeats (CRISPR) depends on the expression of the CRISPR-associated (Cas) proteins and the formation of small CRISPR RNAs (crRNAs). The crRNA-loaded Cas ribonucleoprotein complexes convey the specific recognition and inactivation of target nucleic acids. In E. coli K12, the maturation of crRNAs and the interference with target DNA is performed by the Cascade complex. The transcription of the Cascade operon is tightly repressed through H-NS-dependent inhibition of the Pcas promoter. Elevated levels of the LysR-type regulator LeuO induce the Pcas promoter and concomitantly activate the CRISPR-mediated immunity against phages. Here, we show that the Pcas promoter can also be induced by constitutive expression of the regulator BglJ. This activation is LeuO-dependent as heterodimers of BglJ and RcsB activate leuO transcription. Each transcription factor, LeuO or BglJ, induced the transcription of the Cascade genes to comparable amounts. However, the maturation of the crRNAs was activated in LeuO but not in BglJ-expressing cells. Studies on CRISPR promoter activities, transcript stabilities, crRNA processing and Cascade protein levels were performed to answer the question why crRNA maturation is defective in BglJ-expressing cells. Our results demonstrate that the activation of Cascade gene transcription is necessary but not sufficient to turn on the CRISPR-mediated immunity and suggest a more complex regulation of the type I-E CRISPR-Cas system in E. coli.


Assuntos
Proteínas Associadas a CRISPR/genética , Escherichia coli K12/genética , Proteínas de Escherichia coli/genética , Óperon , RNA Bacteriano/metabolismo , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Associadas a CRISPR/química , Proteínas Associadas a CRISPR/metabolismo , Escherichia coli K12/química , Escherichia coli K12/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Regiões Promotoras Genéticas , RNA Bacteriano/química , RNA Bacteriano/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Transativadores/química , Fatores de Transcrição/química
15.
J Struct Biol ; 178(3): 350-62, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22531577

RESUMO

The prokaryotic immune system, CRISPR, confers an adaptive and inheritable defense mechanism against invasion by mobile genetic elements. Guided by small CRISPR RNAs (crRNAs), a diverse family of CRISPR-associated (Cas) proteins mediates the targeting and inactivation of foreign DNA. Here, we demonstrate that Csn2, a Cas protein likely involved in spacer integration, forms a tetramer in solution and structurally possesses a ring-like structure. Furthermore, co-purified Ca(2+) was found important for the DNA binding property of Csn2, which contains a helicase fold, with highly conserved DxD and RR motifs found throughout Csn2 proteins. We could verify that Csn2 binds ds-DNA. In addition molecular dynamics simulations suggested a Csn2 conformation that can "sit" on the DNA helix and binds DNA in a groove on the outside of the ring.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X/métodos , Streptococcus agalactiae/metabolismo , DNA/metabolismo , Ligação Proteica
16.
Microbiology (Reading) ; 158(Pt 10): 2480-2491, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22767549

RESUMO

6S RNA from Escherichia coli acts as a versatile transcriptional regulator by binding to the RNA polymerase and changing promoter selectivity. Although homologous 6S RNA structures exist in a wide range of bacteria, including cyanobacteria, our knowledge of 6S RNA function results almost exclusively from studies with E. coli. To test for potential structural and functional conservation, we selected four predicted cyanobacterial 6S RNAs (Synechocystis, Synechococcus, Prochlorococcus and Nostoc), which we compared with their E. coli counterpart. Temperature-gradient gel electrophoresis revealed similar thermodynamic transition profiles for all 6S RNAs, indicating basically similar secondary structures. Subtle differences in melting behaviour of the different RNAs point to minor structural variations possibly linked to differences in optimal growth temperature. Secondary structural analysis of three cyanobacterial 6S RNAs employing limited enzymic hydrolysis and in-line probing supported the predicted high degree of secondary structure conservation. Testing for functional homology we found that all cyanobacterial 6S RNAs were active in binding E. coli RNA polymerase and transcriptional inhibition, and had the ability to act as template for transcription of product RNAs (pRNAs). Deletion of the 6S RNA gene in Synechocystis did not significantly affect cell growth in liquid media but reduced fitness during growth on solid agar. While our study shows that basic 6S RNA functions are conserved in species as distantly related as E. coli and cyanobacteria, we also noted a subtle degree of divergence, which might reflect fundamental differences in transcriptional regulation and lifestyle, thus providing the first evidence for a possible physiological role in cyanobacteria.


Assuntos
Cianobactérias/genética , RNA Bacteriano/genética , Cianobactérias/classificação , Cianobactérias/crescimento & desenvolvimento , Cianobactérias/metabolismo , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Nostoc/genética , Nostoc/metabolismo , Prochlorococcus/genética , Prochlorococcus/metabolismo , RNA Bacteriano/metabolismo , RNA não Traduzido , Synechococcus/genética , Synechococcus/metabolismo , Synechocystis/genética , Synechocystis/metabolismo , Transcrição Gênica
17.
Biol Chem ; 393(12): 1513-22, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23667906

RESUMO

6S RNA is a bacterial transcriptional regulator,which accumulates during stationary phase and inhibits transcription from many promoters due to stable association with σ 70 -containing RNA polymerase. This inhibitory RNA polymerase ∼ 6S RNA complex dissociates during nutritional upshift, when cells undergo outgrowth from stationary phase, releasing active RNA polymerase ready for transcription. The release reaction depends on a characteristic property of 6S RNAs, namely to act as template for the de novo synthesis of small RNAs, termed pRNAs.Here, we used limited hydrolysis with structure-specific RNases and in-line probing of isolated 6S RNA and 6SRNA ∼ pRNA complexes to investigate the molecular details leading to the release reaction. Our results indicate that pRNA transcription induces the refolding of the 6S RNA secondary structure by disrupting part of the closing stem(conserved sequence regions CRI and CRIV) and formation of a new hairpin (conserved sequence regions CRIII and CRIV). Comparison of the dimethylsulfate modification pattern of 6S RNA in living cells at stationary growth and during outgrowth confirmed the conformational change observed in vitro. Based on our results, a model describing the individual steps of the release reaction is presented.


Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/enzimologia , RNA Bacteriano/metabolismo , Fator sigma/metabolismo , Sequência de Bases , Escherichia coli/química , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Hidrólise , Dados de Sequência Molecular , Conformação de Ácido Nucleico , RNA Bacteriano/química , RNA Bacteriano/genética , RNA não Traduzido , Transcrição Gênica
18.
Bioorg Med Chem Lett ; 22(11): 3747-50, 2012 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-22542020

RESUMO

Aryl dihydrouracil derivatives were identified from high throughput screening as potent inhibitors of HCV NS5B polymerase. The aryl dihydrouracil derivatives were shown to be non-competitive with respect to template RNA and elongation nucleotide substrates. They demonstrated genotype 1 specific activity towards HCV NS5B polymerases. Structure activity relationships and genotype specific activities of aryl dihydrouracil derivatives suggested that they bind to the palm initiation nucleotide pocket, a hypothesis which was confirmed by studies with polymerases containing mutations in various inhibitor binding sites. Therefore, aryl dihydrouracil derivatives represent a novel class of palm initiation site inhibitors of HCV NS5B polymerase.


Assuntos
Inibidores de Proteases/química , Uracila/análogos & derivados , Proteínas não Estruturais Virais/antagonistas & inibidores , Substituição de Aminoácidos , Genótipo , Hepacivirus/efeitos dos fármacos , Hepacivirus/enzimologia , Cinética , Inibidores de Proteases/síntese química , Inibidores de Proteases/farmacologia , Relação Estrutura-Atividade , Sítio de Iniciação de Transcrição , Uracila/síntese química , Uracila/química , Uracila/farmacologia , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo
19.
Mol Microbiol ; 75(6): 1495-512, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-20132443

RESUMO

Inheritable bacterial defence systems against phage infection and foreign DNA, termed CRISPR (clustered regularly interspaced short palindromic repeats), consist of cas protein genes and repeat arrays interspaced with sequences originating from invaders. The Cas proteins together with processed small spacer-repeat transcripts (crRNAs) cause degradation of penetrated foreign DNA by unknown mechanisms. Here, we have characterized previously unidentified promoters of the Escherichia coli CRISPR arrays and cas protein genes. Transcription of precursor crRNA is directed by a promoter located within the CRISPR leader. A second promoter, directing cas gene transcription, is located upstream of the genes encoding proteins of the Cascade complex. Furthermore, we demonstrate that the DNA-binding protein H-NS is involved in silencing the CRISPR-cas promoters, resulting in cryptic Cas protein expression. Our results demonstrate an active involvement of H-NS in the induction of the CRISPR-cas system and suggest a potential link between two prokaryotic defence systems against foreign DNA.


Assuntos
Proteínas de Escherichia coli/biossíntese , Escherichia coli/fisiologia , Proteínas de Fímbrias/metabolismo , Regulação Bacteriana da Expressão Gênica , Inativação Gênica , Sequências Repetidas Invertidas , Regiões Promotoras Genéticas , Sequência de Bases , DNA/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Ordem dos Genes , Modelos Biológicos , Dados de Sequência Molecular , Família Multigênica , RNA Bacteriano/biossíntese , Sítio de Iniciação de Transcrição , Transcrição Gênica
20.
Mol Microbiol ; 77(6): 1380-93, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20659289

RESUMO

The recently discovered prokaryotic CRISPR/Cas defence system provides immunity against viral infections and plasmid conjugation. It has been demonstrated that in Escherichia coli transcription of the Cascade genes (casABCDE) and to some extent the CRISPR array is repressed by heat-stable nucleoid-structuring (H-NS) protein, a global transcriptional repressor. Here we elaborate on the control of the E. coli CRISPR/Cas system, and study the effect on CRISPR-based anti-viral immunity. Transformation of wild-type E. coli K12 with CRISPR spacers that are complementary to phage Lambda does not lead to detectable protection against Lambda infection. However, when an H-NS mutant of E. coli K12 is transformed with the same anti-Lambda CRISPR, this does result in reduced sensitivity to phage infection. In addition, it is demonstrated that LeuO, a LysR-type transcription factor, binds to two sites flanking the casA promoter and the H-NS nucleation site, resulting in derepression of casABCDE12 transcription. Overexpression of LeuO in E. coli K12 containing an anti-Lambda CRISPR leads to an enhanced protection against phage infection. This study demonstrates that in E. coli H-NS and LeuO are antagonistic regulators of CRISPR-based immunity.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/metabolismo , Escherichia coli K12/genética , Escherichia coli K12/imunologia , Proteínas de Escherichia coli/genética , Fatores de Transcrição/genética , Bacteriófago lambda/fisiologia , Sequência de Bases , Clonagem Molecular , Pegada de DNA , DNA Bacteriano/genética , DNA Intergênico/genética , Escherichia coli K12/virologia , Regulação Bacteriana da Expressão Gênica , Dados de Sequência Molecular , Mutação , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas , Transcrição Gênica
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