RESUMO
Rho is a ring-shaped hexameric ATP-dependent molecular motor. Together with the transcription elongation factor NusG, Rho mediates factor-dependent transcription termination and transcription-translation-coupling quality control in Escherichia coli1-4. Here we report the preparation of complexes that are functional in factor-dependent transcription termination from Rho, NusG, RNA polymerase (RNAP), and synthetic nucleic acid scaffolds, and we report cryogenic electron microscopy structures of the complexes. The structures show that functional factor-dependent pre-termination complexes contain a closed-ring Rho hexamer; have RNA threaded through the central channel of Rho; have 60 nucleotides of RNA interacting sequence-specifically with the exterior of Rho and 6 nucleotides of RNA interacting sequence-specifically with the central channel of Rho; have Rho oriented relative to RNAP such that ATP-dependent translocation by Rho exerts mechanical force on RNAP; and have NusG bridging Rho and RNAP. The results explain five decades of research on Rho and provide a foundation for understanding Rho's function.
Assuntos
Proteínas de Escherichia coli , Escherichia coli , Fatores de Transcrição , Terminação da Transcrição Genética , Trifosfato de Adenosina/metabolismo , Microscopia Crioeletrônica , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/metabolismo , RNA Polimerases Dirigidas por DNA/ultraestrutura , Escherichia coli/química , Escherichia coli/enzimologia , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/ultraestrutura , RNA/química , RNA/genética , RNA/metabolismo , RNA/ultraestrutura , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Fatores de Transcrição/ultraestruturaRESUMO
DksA and ppGpp are the central players in the stringent response and mediate a complete reprogramming of the transcriptome. A major component of the response is a reduction in ribosome synthesis, which is accomplished by the synergistic action of DksA and ppGpp bound to RNA polymerase (RNAP) inhibiting transcription of rRNAs. Here, we report the X-ray crystal structures of Escherichia coli RNAP in complex with DksA alone and with ppGpp. The structures show that DksA accesses the template strand at the active site and the downstream DNA binding site of RNAP simultaneously and reveal that binding of the allosteric effector ppGpp reshapes the RNAP-DksA complex. The structural data support a model for transcriptional inhibition in which ppGpp potentiates the destabilization of open complexes by DksA. This work establishes a structural basis for understanding the pleiotropic effects of DksA and ppGpp on transcriptional regulation in proteobacteria.
Assuntos
Proteínas de Escherichia coli/química , Escherichia coli/química , Nucleotídeos de Guanina/química , Modelos Químicos , Modelos Moleculares , Regulação Alostérica , Domínio Catalítico , Cristalografia por Raios X , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Nucleotídeos de Guanina/metabolismo , Transcriptoma/fisiologiaRESUMO
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/genéticaRESUMO
In σ-dependent transcriptional pausing, the transcription initiation factor σ, translocating with RNA polymerase (RNAP), makes sequence-specific proteinDNA interactions with a promoter-like sequence element in the transcribed region, inducing pausing. It has been proposed that, in σ-dependent pausing, the RNAP active center can access off-pathway "backtracked" states that are substrates for the transcript-cleavage factors of the Gre family and on-pathway "scrunched" states that mediate pause escape. Here, using site-specific proteinDNA photocrosslinking to define positions of the RNAP trailing and leading edges and of σ relative to DNA at the λPR' promoter, we show directly that σ-dependent pausing in the absence of GreB in vitro predominantly involves a state backtracked by 24 bp, and σ-dependent pausing in the presence of GreB in vitro and in vivo predominantly involves a state scrunched by 23 bp. Analogous experiments with a library of 47 (â¼16,000) transcribed-region sequences show that the state scrunched by 23 bpand only that stateis associated with the consensus sequence, T−3N−2Y−1G+1, (where −1 corresponds to the position of the RNA 3' end), which is identical to the consensus for pausing in initial transcription and which is related to the consensus for pausing in transcription elongation. Experiments with heteroduplex templates show that sequence information at position T−3 resides in the DNA nontemplate strand. A cryoelectron microscopy structure of a complex engaged in σ-dependent pausing reveals positions of DNA scrunching on the DNA nontemplate and template strands and suggests that position T−3 of the consensus sequence exerts its effects by facilitating scrunching.
Assuntos
RNA Polimerases Dirigidas por DNA , Transcrição Gênica , Microscopia Crioeletrônica , DNA , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/genéticaRESUMO
All organisms-bacteria, archaea, and eukaryotes-have a transcription initiation factor that contains a structural module that binds within the RNA polymerase (RNAP) active-center cleft and interacts with template-strand single-stranded DNA (ssDNA) in the immediate vicinity of the RNAP active center. This transcription initiation-factor structural module preorganizes template-strand ssDNA to engage the RNAP active center, thereby facilitating binding of initiating nucleotides and enabling transcription initiation from initiating mononucleotides. However, this transcription initiation-factor structural module occupies the path of nascent RNA and thus presumably must be displaced before or during initial transcription. Here, we report four sets of crystal structures of bacterial initially transcribing complexes that demonstrate and define details of stepwise, RNA-extension-driven displacement of the "σ-finger" of the bacterial transcription initiation factor σ. The structures reveal that-for both the primary σ-factor and extracytoplasmic (ECF) σ-factors, and for both 5'-triphosphate RNA and 5'-hydroxy RNA-the "σ-finger" is displaced in stepwise fashion, progressively folding back upon itself, driven by collision with the RNA 5'-end, upon extension of nascent RNA from â¼5 nt to â¼10 nt.
Assuntos
Proteínas de Escherichia coli/química , Fator sigma/química , Iniciação da Transcrição Genética , Sítios de Ligação , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli , Proteínas de Escherichia coli/metabolismo , Simulação de Dinâmica Molecular , Regiões Promotoras Genéticas , Ligação Proteica , RNA/química , RNA/genética , RNA/metabolismo , Fator sigma/metabolismoRESUMO
After transcription termination, cellular RNA polymerases (RNAPs) are occasionally trapped on DNA, impounded in an undefined post-termination complex (PTC), limiting the free RNAP pool and subsequently leading to inefficient transcription. In Escherichia coli, a Swi2/Snf2 family of ATPase called RapA is known to be involved in countering such inefficiency through RNAP recycling; however, the precise mechanism of this recycling is unclear. To better understand its mechanism, here we determined the structures of two sets of E. coli RapA-RNAP complexes, along with the RNAP core enzyme and the elongation complex, using cryo-EM. These structures revealed the large conformational changes of RNAP and RapA upon their association that has been implicated in the hindrance of PTC formation. Our results along with DNA-binding assays reveal that although RapA binds RNAP away from the DNA-binding main channel, its binding can allosterically close the RNAP clamp, thereby preventing its nonspecific DNA binding and PTC formation. Taken together, we propose that RapA acts as a guardian of RNAP by which RapA prevents nonspecific DNA binding of RNAP without affecting the binding of promoter DNA recognition σ factor, thereby enhancing RNAP recycling.
Assuntos
Adenosina Trifosfatases/química , DNA Bacteriano/química , RNA Polimerases Dirigidas por DNA/química , Proteínas de Escherichia coli/química , Escherichia coli/química , Complexos Multienzimáticos/química , Adenosina Trifosfatases/metabolismo , Microscopia Crioeletrônica , DNA Bacteriano/metabolismo , DNA Bacteriano/ultraestrutura , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/metabolismo , Escherichia coli/ultraestrutura , Proteínas de Escherichia coli/metabolismo , Complexos Multienzimáticos/metabolismo , Complexos Multienzimáticos/ultraestruturaRESUMO
Rifamycin antibiotics are a valuable class of antimicrobials for treating infections by mycobacteria and other persistent bacteria owing to their potent bactericidal activity against replicating and non-replicating pathogens. However, the clinical utility of rifamycins against Mycobacterium abscessus is seriously compromised by a novel resistance mechanism, namely, rifamycin inactivation by ADP-ribosylation. Using a structure-based approach, we rationally redesign rifamycins through strategic modification of the ansa-chain to block ADP-ribosylation while preserving on-target activity. Validated by a combination of biochemical, structural, and microbiological studies, the most potent analogs overcome ADP-ribosylation, restored their intrinsic low nanomolar activity and demonstrated significant in vivo antibacterial efficacy. Further optimization by tuning drug disposition properties afforded a preclinical candidate with remarkable potency and an outstanding pharmacokinetic profile.
Assuntos
Mycobacterium , Rifamicinas , Antibacterianos/farmacologia , Antibacterianos/química , Testes de Sensibilidade Microbiana , Rifamicinas/farmacologia , Rifamicinas/química , ADP-RibosilaçãoRESUMO
Reiterative transcription is a noncanonical form of RNA synthesis in which a nucleotide specified by a single base in the DNA template is repetitively added to the nascent transcript. Here we determined the crystal structure of an RNA polymerase, the bacterial enzyme from Thermus thermophilus, engaged in reiterative transcription during transcription initiation at a promoter resembling the pyrG promoter of Bacillus subtilis The structure reveals that the reiterative transcript detours from the dedicated RNA exit channel and extends toward the main channel of the enzyme, thereby allowing RNA extension without displacement of the promoter recognition σ-factor. Nascent transcripts containing reiteratively added G residues are eventually extended by nonreiterative transcription, revealing an atypical pathway for the formation of a transcription elongation complex.
Assuntos
Proteínas de Bactérias/química , RNA Polimerases Dirigidas por DNA/química , RNA Bacteriano/genética , Thermus thermophilus/genética , Transcrição Gênica , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Modelos Moleculares , Mutação , Poli G , Regiões Promotoras Genéticas , RNA Bacteriano/metabolismo , Rifampina/farmacologia , Thermus thermophilus/enzimologiaRESUMO
Genomes of phages, mitochondria, and chloroplasts are transcribed by a diverse group of transcriptional machineries with structurally related single-subunit RNA polymerases (RNAPs). Our understanding of transcription mechanisms of these enzymes is predominantly based on biochemical and structural studies of three most-studied members, transcription factor-independent phage T7 RNAP, transcription factor-dependent phage N4 virion-encapsidated RNAP, and transcription factor-dependent mitochondrial RNAPs (mtRNAP). Although these RNAPs employ completely different mechanisms for promoter recognition and transcription termination, these enzymes are relatively large and formed by single polypeptides. Historically being a model enzyme for studying the mechanisms of transcription by T7-like RNAPs, however, T7 RNAP represents only a small group of RNAPs in this family. The vast majority of T7-like RNAPs are transcription factor-dependent, and several of them are heterodimeric enzymes. Here, we report X-ray crystal structures of transcription complexes of the smallest and heterodimeric form of T7-like RNAP, bacteriophage N4 RNAPII, providing insights into the structural organization of a minimum RNAP in this family. We analyze structural and functional aspects of heterodimeric architecture of N4 RNAPII concerning the mechanisms of transcription initiation and transition to processive RNA elongation. Interestingly, N4 RNAPII maintains the same conformation in promoter-bound and elongation transcription complexes, revealing a novel transcription mechanism for single-subunit RNAPs. This work establishes a structural basis for studying mechanistic aspects of transcription by factor-dependent minimum RNAP.
Assuntos
Bacteriófago N4/química , RNA Polimerase II/química , Proteínas Virais/química , Bacteriófago N4/genética , Bacteriófago N4/metabolismo , Cristalografia por Raios X , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Simulação de Acoplamento Molecular , Regiões Promotoras Genéticas , Ligação Proteica , Conformação Proteica , Multimerização Proteica , RNA Polimerase II/metabolismo , Ativação Transcricional , Proteínas Virais/metabolismoRESUMO
Since 1967, Rifampin (RMP, a Rifamycin) has been used as a first line antibiotic treatment for tuberculosis (TB), and it remains the cornerstone of current short-term TB treatment. Increased occurrence of Rifamycin-resistant (RIFR ) TB, â¼41% of which results from the RpoB S531L mutation in RNA polymerase (RNAP), has become a growing problem worldwide. In this study, we determined the X-ray crystal structures of the Escherichia coli RNAPs containing the most clinically important S531L mutation and two other frequently observed RIFR mutants, RpoB D516V and RpoB H526Y. The structures reveal that the S531L mutation imparts subtle if any structural or functional impact on RNAP in the absence of RIF. However, upon RMP binding, the S531L mutant exhibits a disordering of the RIF binding interface, which effectively reduces the RMP affinity. In contrast, the H526Y mutation reshapes the RIF binding pocket, generating significant steric conflicts that essentially prevent any RIF binding. While the D516V mutant does not exhibit any such gross structural changes, certainly the electrostatic surface of the RIF binding pocket is dramatically changed, likely resulting in the decreased affinity for RIFs. Analysis of interactions of RMP with three common RIFR mutant RNAPs suggests that modifications to RMP may recover its efficacy against RIFR TB.
Assuntos
Antibióticos Antituberculose/farmacologia , Proteínas de Bactérias/genética , RNA Polimerases Dirigidas por DNA/ultraestrutura , Mycobacterium tuberculosis/efeitos dos fármacos , Rifampina/farmacologia , Tuberculose Pulmonar/tratamento farmacológico , Sítios de Ligação/genética , Cristalografia por Raios X , RNA Polimerases Dirigidas por DNA/efeitos dos fármacos , RNA Polimerases Dirigidas por DNA/genética , Farmacorresistência Bacteriana/genética , Escherichia coli/genética , Humanos , Mutação/genética , Mycobacterium tuberculosis/genética , Conformação Proteica , RNA Bacteriano , Tuberculose Pulmonar/microbiologiaRESUMO
The bacterial RNA polymerase (RNAP) holoenzyme containing σ factor initiates transcription at specific promoter sites by de novo RNA priming, the first step of RNA synthesis where RNAP accepts two initiating ribonucleoside triphosphates (iNTPs) and performs the first phosphodiester bond formation. We present the structure of de novo transcription initiation complex that reveals unique contacts of the iNTPs bound at the transcription start site with the template DNA and also with RNAP and demonstrate the importance of these contacts for transcription initiation. To get further insight into the mechanism of RNA priming, we determined the structure of initially transcribing complex of RNAP holoenzyme with 6-mer RNA, obtained by in crystallo transcription approach. The structure highlights RNAP-RNA contacts that stabilize the short RNA transcript in the active site and demonstrates that the RNA 5'-end displaces σ region 3.2 from its position near the active site, which likely plays a key role in σ ejection during the initiation-to-elongation transition. Given the structural conservation of the RNAP active site, the mechanism of de novo RNA priming appears to be conserved in all cellular RNAPs.
Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , Sítio de Iniciação de Transcrição , Sequência de Bases , Sítios de Ligação , Cristalização , Cristalografia por Raios X , DNA Bacteriano/genética , RNA Polimerases Dirigidas por DNA/química , Modelos Moleculares , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Homologia de Sequência do Ácido Nucleico , Thermus thermophilus/enzimologiaRESUMO
Structures recently have been reported of molecular assemblies that mediate transcription-translation coupling in Escherichia coli . In these molecular assemblies, termed "coupled transcription-translation complexes" or "TTC-B", RNA polymerase (RNAP) interacts directly with the ribosome, the transcription elongation factor NusG or its paralog RfaH forms a bridge between RNAP and ribosome, and the transcription elongation factor NusA optionally forms a second bridge between RNAP and ribosome. Here, we have determined structures of coupled transcription-translation complexes having mRNA spacers between RNAP and ribosome longer than the maximum-length mRNA spacer compatible with formation of TTC-B. The results define a new class of coupled transcription-translation complex, termed "TTC-LC," where "LC" denotes "long-range coupling." TTC-LC differs from TTC-B by a â¼60° rotation and â¼70 Å translation of RNAP relative to ribosome, resulting in loss of direct interactions between RNAP and ribosome and creation of a â¼70 Å gap between RNAP and ribosome. TTC-LC accommodates long mRNA spacers by looping out mRNA from the gap between RNAP and ribosome. We propose that TTC-LC is an intermediate in assembling and disassembling TTC-B, mediating pre-TTC-B transcription-translation coupling before a ribosome catches up to RNAP, and mediating post-TTC-B transcription-translation coupling after a ribosome stops moving and RNAP continues moving.
RESUMO
The NusG paralog RfaH mediates bacterial transcription-translation coupling in genes that contain a DNA sequence element, termed an ops site, required for pausing RNA polymerase (RNAP) and for loading RfaH onto the paused RNAP. Here, we report cryo-electron microscopy structures of transcription-translation complexes (TTCs) containing Escherichia coli RfaH. The results show that RfaH bridges RNAP and the ribosome, with the RfaH N-terminal domain interacting with RNAP and the RfaH C-terminal domain interacting with the ribosome. The results show that the distribution of translational and orientational positions of RNAP relative to the ribosome in RfaH-coupled TTCs is more restricted than in NusG-coupled TTCs because of the more restricted flexibility of the RfaH interdomain linker. The results further suggest that the structural organization of RfaH-coupled TTCs in the 'loading state', in which RNAP and RfaH are located at the ops site during formation of the TTC, is the same as the structural organization of RfaH-coupled TTCs in the 'loaded state', in which RNAP and RfaH are located at positions downstream of the ops site during function of the TTC. The results define the structural organization of RfaH-containing TTCs and set the stage for analysis of functions of RfaH during translation initiation and transcription-translation coupling.
RESUMO
The NusG paralog RfaH mediates bacterial transcription-translation coupling on genes that contain a DNA sequence element, termed an ops site, required for pausing RNA polymerase (RNAP) and for loading RfaH onto the paused RNAP. Here we report cryo-EM structures of transcription-translation complexes (TTCs) containing RfaH. The results show that RfaH bridges RNAP and the ribosome, with the RfaH N-terminal domain interacting with RNAP, and with the RfaH C-terminal domain interacting with the ribosome. The results show that the distribution of translational and orientational positions of RNAP relative to the ribosome in RfaH-coupled TTCs is more restricted than in NusG-coupled TTCs, due to the more restricted flexibility of the RfaH interdomain linker. The results further show that the structural organization of RfaH-coupled TTCs in the "loading state," in which RNAP and RfaH are located at the ops site during formation of the TTC, is the same as the structural organization of RfaH-coupled TTCs in the "loaded state," in which RNAP and RfaH are located at positions downstream of the ops site during function of the TTC. The results define the structural organization of RfaH-containing TTCs and set the stage for analysis of functions of RfaH during translation initiation and transcription-translation coupling. One sentence summary: Cryo-EM reveals the structural basis of transcription-translation coupling by RfaH.
RESUMO
The ribonuclease FttA mediates factor-dependent transcription termination in archaea 1-3 . Here, we report the structure of a Thermococcus kodakarensis transcription pre-termination complex comprising FttA, Spt4, Spt5, and a transcription elongation complex (TEC). The structure shows that FttA interacts with the TEC in a manner that enables RNA to proceed directly from the TEC RNA-exit channel to the FttA catalytic center and that enables endonucleolytic cleavage of RNA by FttA, followed by 5'â3' exonucleolytic cleavage of RNA by FttA and concomitant 5'â3' translocation of FttA on RNA, to apply mechanical force to the TEC and trigger termination. The structure further reveals that Spt5 bridges FttA and the TEC, explaining how Spt5 stimulates FttA-dependent termination. The results reveal functional analogy between bacterial and archaeal factor-dependent termination, reveal functional homology between archaeal and eukaryotic factor-dependent termination, and reveal fundamental mechanistic similarities in factor-dependent termination in the three domains of life: bacterial, archaeal, and eukaryotic. One sentence summary: Cryo-EM reveals the structure of the archaeal FttA pre-termination complex.
RESUMO
TNP-2198, a stable conjugate of a rifamycin pharmacophore and a nitroimidazole pharmacophore, has been designed, synthesized, and evaluated as a novel dual-targeted antibacterial agent for the treatment of microaerophilic and anaerobic bacterial infections. TNP-2198 exhibits greater activity than a 1:1 molar mixture of the parent drugs and exhibits activity against strains resistant to both rifamycins and nitroimidazoles. A crystal structure of TNP-2198 bound to a Mycobacterium tuberculosis RNA polymerase transcription initiation complex reveals that the rifamycin portion of TNP-2198 binds to the rifamycin binding site on RNAP and the nitroimidazole portion of TNP-2198 interacts directly with the DNA template-strand in the RNAP active-center cleft, forming a hydrogen bond with a base of the DNA template strand. TNP-2198 is currently in Phase 2 clinical development for the treatment of Helicobacter pylori infection, Clostridioides difficile infection, and bacterial vaginosis.
Assuntos
Infecções por Helicobacter , Helicobacter pylori , Nitroimidazóis , Rifamicinas , Anaerobiose , RNA Polimerases Dirigidas por DNA , Humanos , Nitroimidazóis/farmacologiaRESUMO
In bacteria, transcription and translation are coupled processes in which the movement of RNA polymerase (RNAP)-synthesizing messenger RNA (mRNA) is coordinated with the movement of the first ribosome-translating mRNA. Coupling is modulated by the transcription factors NusG (which is thought to bridge RNAP and the ribosome) and NusA. Here, we report cryo-electron microscopy structures of Escherichia coli transcription-translation complexes (TTCs) containing different-length mRNA spacers between RNAP and the ribosome active-center P site. Structures of TTCs containing short spacers show a state incompatible with NusG bridging and NusA binding (TTC-A, previously termed "expressome"). Structures of TTCs containing longer spacers reveal a new state compatible with NusG bridging and NusA binding (TTC-B) and reveal how NusG bridges and NusA binds. We propose that TTC-B mediates NusG- and NusA-dependent transcription-translation coupling.
Assuntos
RNA Polimerases Dirigidas por DNA/química , Proteínas de Escherichia coli/química , Escherichia coli/genética , Fatores de Alongamento de Peptídeos/química , Biossíntese de Proteínas , Fatores de Transcrição/química , Transcrição Gênica , Fatores de Elongação da Transcrição/química , Microscopia Crioeletrônica , Regulação Bacteriana da Expressão Gênica , Ligação Proteica , Conformação Proteica , RNA Mensageiro/químicaRESUMO
Chronic wounds infected with pathogens such as Staphylococcus aureus represent a worldwide health concern, especially in patients with a compromised immune system. As antimicrobial resistance has become an immense global problem, novel antibiotics are urgently needed. One strategy to overcome this threatening situation is the search for drugs targeting novel binding sites on essential and validated enzymes such as the bacterial RNA polymerase (RNAP). In this work, we describe the establishment of an in vivo wound infection model based on the pathogen S. aureus and hairless Crl:SKH1-Hrhr (SKH1) mice. The model proved to be a valuable preclinical tool to study selected RNAP inhibitors after topical application. While rifampicin showed a reduction in the loss of body weight induced by the bacteria, an acceleration of wound healing kinetics, and a reduced number of colony forming units in the wound, the ureidothiophene-2-carboxylic acid 1 was inactive under in vivo conditions, probably due to strong plasma protein binding. The cocrystal structure of compound 1 with RNAP, that we hereby also present, will be of great value for applying appropriate structural modifications to further optimize the compound, especially in terms of plasma protein binding.
Assuntos
Infecções Estafilocócicas , Infecção dos Ferimentos , Animais , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , RNA Polimerases Dirigidas por DNA , Humanos , Camundongos , Infecções Estafilocócicas/tratamento farmacológico , Staphylococcus aureus , Infecção dos Ferimentos/tratamento farmacológicoRESUMO
Precise regulation of gene expression is crucial for bacteria to respond to changing environmental conditions. In addition to protein factors affecting RNA polymerase (RNAP) activity, second messengers play an important role in transcription regulation, such as well-known effectors of the stringent response: guanosine 5'triphosphate-3'diphosphate and guanosine 3', 5'-bis(diphosphate) [(p)ppGpp]. Although much is known about importance of the 5' and 3' moieties of (p)ppGpp, the role of the guanine base remains somewhat cryptic. Here, we use (p)ppGpp's adenine analogs [(p)ppApp] to investigate how the nucleobase contributes to determine its binding site and transcriptional regulation. We determined X-ray crystal structure of Escherichia coli RNAP-(p)ppApp complex, which shows the analogs bind near the active site and switch regions of RNAP. We have also explored the regulatory effects of (p)ppApp on transcription initiating from the well-studied E. coli rrnB P1 promoter to assess and compare properties of (p)ppApp with (p)ppGpp. We demonstrate that contrary to (p)ppGpp, (p)ppApp activates transcription at this promoter and DksA hinders this effect. Moreover, pppApp exerts a stronger effect than ppApp. We also show that when ppGpp and pppApp are present together, the outcome depends on which one of them was pre-incubated with RNAP first. This behavior suggests a surprising Yin-Yang like reciprocal plasticity of RNAP responses at a single promoter, occasioned simply by pre-exposure to one or the other nucleotide. Our observations underscore the importance of the (p)ppNpp's purine nucleobase for interactions with RNAP, which may lead to a better fundamental understanding of (p)ppGpp regulation of RNAP activity.
Assuntos
Nucleotídeos de Adenina/química , RNA Polimerases Dirigidas por DNA/química , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Regiões Promotoras Genéticas , Ativação Transcricional , Nucleotídeos de Adenina/metabolismo , Sítios de Ligação , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/enzimologia , Proteínas de Escherichia coli/metabolismo , Guanosina Pentafosfato/química , Guanosina Pentafosfato/metabolismo , Modelos Moleculares , Relação Estrutura-AtividadeRESUMO
Rifampin has been a cornerstone of tuberculosis (TB) treatment since its introduction. The rise of multidrug-resistant and extensively drug-resistant TB makes the development of novel therapeutics effective against these strains an urgent need. Site-specific mutations in the target enzyme of rifampin, RNA polymerase (RNAP) comprises the majority (~97%) of rifamycin-resistant (RifR) strains of Mycobacterium tuberculosis (MTB). To identify novel inhibitors of bacterial RNAP, an in vitro plasmid-based transcription assay that uses malachite green (MG) to detect transcribed RNA containing MG aptamers was developed. This assay was optimized in a 384-well plate format and used to screen 150,000 compounds against an Escherichia coli homolog of the most clinically relevant RifR RNAP (ßS531L) containing a mutation (ß'V408G) that compensates for the fitness defect of this RifR mutant. Following confirmation and concentration-response studies, 10 compounds were identified with similar in vitro inhibition values across a panel of wild-type and RifR E. coli and MTB RNAPs. Four compounds identified from the screen are active against MTB in culture at concentrations below 200 µM. Initial follow-up has resulted in the elimination of one scaffold due to potential pan-assay interference.