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When analyzing cancer sample genomes in clinical practice, many structural variants (SVs), other than single nucleotide variants (SNVs), have been identified. To identify driver variants, the leading candidates must be narrowed down. When fusion genes are involved, selection is particularly difficult, and highly accurate predictions from AI is important. Furthermore, we also wanted to determine how the prediction can make more reliable diagnoses. Here, we developed an explainable AI (XAI) suitable for SVs with gene fusions, based on the XAI technology we previously developed for the prediction of SNV pathogenicity. To cope with gene fusion variants, we added new data to the previous knowledge graph for SVs and we improved the algorithm. Its prediction accuracy was as high as that of existing tools. Moreover, our XAI could explain the reasons for these predictions. We used some variant examples to demonstrate that the reasons are plausible in terms of pathogenic basic mechanisms. These results can be seen as a hopeful step toward the future of genomic medicine, where efficient and correct decisions can be made with the support of AI.
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PURPOSE: Several studies present the unsuitability of the tilted-wire method for slice sensitivity profile (SSP) in helical scan. We compared the accuracy for SSP by the tilted-wire averaging method using multiple wire profiles and by the conventional micro-coin method. METHODS: A micro-coin phantom positioned at the center or the off-center was scanned using a 64-detector row CT scanner in different positions where an X-ray tube starts scanning. In the same way, tilted-wire averaging phantoms, approximately 70 mm in diameter, in the shape of a donut, 8 wires tilted from the circumference toward the center, were scanned. Images were reconstructed with a slice thickness of 0.5 mm. RESULTS: The relative errors of full width at half maximum (FWHM) by the tilted-wire averaging method were -0.015 mm to -0.004 mm (-1.98% to -0.56%) at the center compared to those by the micro-coin method, and it is almost the same value regardless of the number of wires. Relative errors were 0.001 mm to 0.029 mm (0.11% to 3.74%) at the upper 8 cm from the center, and 0.014 mm to 0.078 mm (1.86% to 10.25%) at the upper 16 cm, and the value of relative errors increased as it got farther from the center and as the number of wires went fewer. CONCLUSION: This study indicated that accurate measurement of SSP may be achieved by using 4 (arranged every 90 degrees) or more averaging wires.
Assuntos
Imagens de Fantasmas , Tomografia Computadorizada por Raios X/métodos , Processamento de Imagem Assistida por Computador/métodosRESUMO
Cyanobacteria and evolutionarily related chloroplasts of algae and plants possess unique RNA polymerases (RNAPs) with characteristics that distinguish them from canonical bacterial RNAPs. The largest subunit of cyanobacterial RNAP (cyRNAP) is divided into two polypeptides, ß'1 and ß'2, and contains the largest known lineage-specific insertion domain, Si3, located in the middle of the trigger loop and spanning approximately half of the ß'2 subunit. In this study, we present the X-ray crystal structure of Si3 and the cryo-EM structures of the cyRNAP transcription elongation complex plus the NusG factor with and without incoming nucleoside triphosphate (iNTP) bound at the active site. Si3 has a well-ordered and elongated shape that exceeds the length of the main body of cyRNAP, fits into cavities of cyRNAP in the absence of iNTP bound at the active site and shields the binding site of secondary channel-binding proteins such as Gre and DksA. A small transition from the trigger loop to the trigger helix upon iNTP binding results in a large swing motion of Si3; however, this transition does not affect the catalytic activity of cyRNAP due to its minimal contact with cyRNAP, NusG, or DNA. This study provides a structural framework for understanding the evolutionary significance of these features unique to cyRNAP and chloroplast RNAP and may provide insights into the molecular mechanism of transcription in specific environment of photosynthetic organisms and organelle.
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Cianobactérias , Proteínas de Escherichia coli , Transcrição Gênica , Escherichia coli/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Cianobactérias/genética , Cianobactérias/metabolismo , DNA/metabolismo , Fatores de Alongamento de Peptídeos/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Escherichia coli/metabolismoRESUMO
Cyanobacteria and evolutionarily related chloroplasts of algae and plants possess unique RNA polymerases (RNAPs) with characteristics that distinguish from canonical bacterial RNAPs. The largest subunit of cyanobacterial RNAP (cyRNAP) is divided into two polypeptides, ß'1 and ß'2, and contains the largest known lineage-specific insertion domain, Si3, located in the middle of the trigger loop and spans approximately half of the ß'2 subunit. In this study, we present the X-ray crystal structure of Si3 and the cryo-EM structures of the cyRNAP transcription elongation complex plus the NusG factor with and without incoming nucleoside triphosphate (iNTP) bound at the active site. Si3 has a well-ordered and elongated shape that exceeds the length of the main body of cyRNAP, fits into cavities of cyRNAP and shields the binding site of secondary channel-binding proteins such as Gre and DksA. A small transition from the trigger loop to the trigger helix upon iNTP binding at the active site results in a large swing motion of Si3; however, this transition does not affect the catalytic activity of cyRNAP due to its minimal contact with cyRNAP, NusG or DNA. This study provides a structural framework for understanding the evolutionary significance of these features unique to cyRNAP and chloroplast RNAP and may provide insights into the molecular mechanism of transcription in specific environment of photosynthetic organisms.
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Hydrothermal systems form at divergent and convergent boundaries of lithospheric plates and within plates due to weakened crust and mantle plumes, playing host to diverse microbial ecosystems. Little is known of how differences in tectonic setting influence the geochemical and microbial compositions of these hydrothermal ecosystems. Here, coordinated geochemical and microbial community analyses were conducted on 87 high-temperature (>65°C) water and sediment samples from hot springs in Yellowstone National Park, Wyoming, USA (n = 41; mantle plume setting), Iceland (n = 41, divergent boundary), and Japan (n = 5; convergent boundary). Region-specific variation in geochemistry and sediment-associated 16S rRNA gene amplicon sequence variant (ASV) composition was observed, with 16S rRNA gene assemblages being nearly completely distinguished by region and pH being the most explanatory parameter within regions. Several low abundance ASVs exhibited cosmopolitan distributions across regions, while most high-abundance ASVs were only identified in specific regions. The presence of some cosmopolitan ASVs across regions argues against dispersal limitation primarily shaping the distribution of taxa among regions. Rather, the results point to local tectonic and geologic characteristics shaping the geochemistry of continental hydrothermal systems that then select for distinct microbial assemblages. These results provide new insights into the co-evolution of hydrothermal systems and their microbial communities.
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Fontes Termais , Microbiota , Fontes Termais/química , RNA Ribossômico 16S/genética , Água , Japão , FilogeniaRESUMO
The processing of the Coronavirus polyproteins pp1a and pp1ab by the main protease Mpro to produce mature proteins is a crucial event in virus replication and a promising target for antiviral drug development. Mpro cleaves polyproteins in a defined order, but how Mpro and/or the polyproteins determine the order of cleavage remains enigmatic due to a lack of structural information about polyprotein-bound Mpro. Here, we present the cryo-EM structures of SARS-CoV-2 Mpro in an apo form and in complex with the nsp7-10 region of the pp1a polyprotein. The complex structure shows that Mpro interacts with only the recognition site residues between nsp9 and nsp10, without any association with the rest of the polyprotein. Comparison between the apo form and polyprotein-bound structures of Mpro highlights the flexible nature of the active site region of Mpro, which allows it to accommodate ten recognition sites found in the polyprotein. These observations suggest that the role of Mpro in selecting a preferred cleavage site is limited and underscores the roles of the structure, conformation, and/or dynamics of the polyproteins in determining the sequence of polyprotein cleavage by Mpro.
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Proteases 3C de Coronavírus , Poliproteínas , Proteólise , SARS-CoV-2 , Humanos , Poliproteínas/metabolismo , SARS-CoV-2/metabolismo , Proteases 3C de Coronavírus/metabolismoRESUMO
NusG is a transcription elongation factor that stimulates transcription pausing in Gram+ bacteria including B. subtilis by sequence-specific interaction with a conserved pause-inducing -11TTNTTT-6 motif found in the non-template DNA (ntDNA) strand within the transcription bubble. To reveal the structural basis of NusG-dependent pausing, we determined a cryo-EM structure of a paused transcription complex (PTC) containing RNA polymerase (RNAP), NusG, and the TTNTTT motif in the ntDNA strand. The interaction of NusG with the ntDNA strand rearranges the transcription bubble by positioning three consecutive T residues in a cleft between NusG and the ß-lobe domain of RNAP. We revealed that the RNAP swivel module rotation (swiveling), which widens (swiveled state) and narrows (non-swiveled state) a cleft between NusG and the ß-lobe, is an intrinsic motion of RNAP and is directly linked to trigger loop (TL) folding, an essential conformational change of all cellular RNAPs for the RNA synthesis reaction. We also determined cryo-EM structures of RNAP escaping from the paused transcription state. These structures revealed the NusG-dependent pausing mechanism by which NusG-ntDNA interaction inhibits the transition from swiveled to non-swiveled states, thereby preventing TL folding and RNA synthesis allosterically. This motion is also reduced by the formation of an RNA hairpin within the RNA exit channel. Thus, the pause half-life can be modulated by the strength of the NusG-ntDNA interaction and/or the stability of the RNA hairpin. NusG residues that interact with the TTNTTT motif are widely conserved in bacteria, suggesting that NusG-dependent pausing is widespread.
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Proteínas de Escherichia coli , Transcrição Gênica , Fatores de Transcrição/genética , RNA Polimerases Dirigidas por DNA/metabolismo , DNA , Bactérias/metabolismo , RNA , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/químicaRESUMO
The transcriptome-wide contributions of Rho-dependent and intrinsic (Rho-independent) transcription termination mechanisms in bacteria are unclear. By sequencing released transcripts in a wild-type strain and strains containing deficiencies in NusA, NusG and/or Rho (10 strains), we produced an atlas of terminators for the model Gram-positive bacterium Bacillus subtilis. We found that NusA and NusG stimulate 77% and 19% of all intrinsic terminators, respectively, and that both proteins participate in Rho-dependent termination. We also show that Rho stimulates termination at 10% of the intrinsic terminators in vivo. We recapitulated Rho-stimulated intrinsic termination at 5 terminators in vitro and found that Rho requires the KOW domain of NusG to stimulate this process at one of these terminators. Computational analyses of our atlas using RNAstructure, MEME suite and DiffLogo, combined with in vitro transcription experiments, revealed that Rho stimulates intrinsic terminators with weak hairpins and/or U-rich tracts by remodelling the RNA upstream of the intrinsic terminator to prevent the formation of RNA structures that could otherwise compete with the terminator hairpin. We also identified 56 putative examples of 'hybrid Rho-dependent termination', wherein classical Rho-dependent termination occurs after readthrough of a Rho-stimulated intrinsic terminator.
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Bacillus subtilis , Transcrição Gênica , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , RNA/metabolismoRESUMO
Argonaute proteins are programmable nucleases that have defense and regulatory functions in both eukaryotes and prokaryotes. All known prokaryotic Argonautes (pAgos) characterized so far act on DNA targets. Here, we describe a new class of pAgos that uniquely use DNA guides to process RNA targets. The biochemical and structural analysis of Pseudooceanicola lipolyticus pAgo (PliAgo) reveals an unusual organization of the guide binding pocket that does not rely on divalent cations and the canonical set of contacts for 5'-end interactions. Unconventional interactions of PliAgo with the 5'-phosphate of guide DNA define its new position within pAgo and shift the site of target RNA cleavage in comparison with known Argonautes. The specificity for RNA over DNA is defined by ribonucleotide residues at the cleavage site. The analysed pAgos sense mismatches and modifications in the RNA target. The results broaden our understanding of prokaryotic defense systems and extend the spectrum of programmable nucleases with potential use in RNA technology.
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Proteínas Argonautas , RNA , Proteínas Argonautas/metabolismo , Bactérias/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , DNA/metabolismo , Endonucleases/metabolismo , Células Procarióticas/metabolismo , RNA/metabolismo , RNA Guia de Cinetoplastídeos/genética , RNA Guia de Cinetoplastídeos/metabolismoRESUMO
Transcription must be properly regulated to ensure dynamic gene expression underlying growth, development, and response to environmental cues. Regulation is imposed throughout the transcription cycle, and while many efforts have detailed the regulation of transcription initiation and early elongation, the termination phase of transcription also plays critical roles in regulating gene expression. Transcription termination can be driven by only a few proteins in each domain of life. Detailing the mechanism(s) employed provides insight into the vulnerabilities of transcription elongation complexes (TECs) that permit regulated termination to control expression of many genes and operons. Here, we describe the biochemical activities and crystal structure of the superfamily 2 helicase Eta, one of two known factors capable of disrupting archaeal transcription elongation complexes. Eta retains a twin-translocase core domain common to all superfamily 2 helicases and a well-conserved C terminus wherein individual amino acid substitutions can critically abrogate termination activities. Eta variants that perturb ATPase, helicase, single-stranded DNA and double-stranded DNA translocase and termination activities identify key regions of the C terminus of Eta that, when combined with modeling Eta-TEC interactions, provide a structural model of Eta-mediated termination guided in part by structures of Mfd and the bacterial TEC. The susceptibility of TECs to disruption by termination factors that target the upstream surface of RNA polymerase and potentially drive termination through forward translocation and allosteric mechanisms that favor opening of the clamp to release the encapsulated nucleic acids emerges as a common feature of transcription termination mechanisms.
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Proteínas Arqueais , DNA Helicases , Thermococcus , Fatores de Transcrição , Terminação da Transcrição Genética , Proteínas Arqueais/química , Proteínas Arqueais/metabolismo , Cristalografia , DNA Helicases/química , DNA Helicases/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Domínios Proteicos , Thermococcus/enzimologia , Thermococcus/genética , Fatores de Transcrição/química , Fatores de Transcrição/metabolismoRESUMO
Stalling of the transcription elongation complex formed by DNA, RNA polymerase (RNAP) and RNA presents a serious obstacle to concurrent processes due to the extremely high stability of the DNA-bound polymerase. RapA, known to remove RNAP from DNA in an ATP-dependent fashion, was identified over 50 years ago as an abundant binding partner of RNAP; however, its mechanism of action remains unknown. Here, we use single-molecule magnetic trapping assays to characterize RapA activity and begin to specify its mechanism of action. We first show that stalled RNAP resides on DNA for times on the order of 106 seconds and that increasing positive torque on the DNA reduces this lifetime. Using stalled RNAP as a substrate we show that the RapA protein stimulates dissociation of stalled RNAP from positively supercoiled DNA but not negatively supercoiled DNA. We observe that RapA-dependent RNAP dissociation is torque-sensitive, is inhibited by GreB and depends on RNA length. We propose that stalled RNAP is dislodged from DNA by RapA via backtracking in a supercoiling- and torque-dependent manner, suggesting that RapA's activity on transcribing RNAP in vivo is responsible for resolving conflicts between converging polymerase molecular motors.
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DNA Super-Helicoidal , Proteínas de Escherichia coli/metabolismo , Escherichia coli , DNA Super-Helicoidal/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , RNA/genética , RNA/metabolismo , Transcrição GênicaRESUMO
Tuberculosis (TB) is one of the most significant world health problems, responsible for 1.5 M deaths in 2020, and yet, current treatments rely largely on 40 year old paradigms. Although the rifamycins (RIFs), best exemplified by the drug rifampin (RMP), represent a well-studied and therapeutically effective chemotype that targets the bacterial RNA polymerase (RNAP), these agents still suffer from serious drawbacks including the following: 3-9 month treatment times; cytochrome P450 (Cyp450) induction [particularly problematic for human immunodeficiency virus-Mycobacterium tuberculosis (MTB) co-infection]; and the existence of RIF-resistant (RIFR) MTB strains. We have utilized a structure-based drug design approach to synthesize and test 15 benzoxazinorifamycins (bxRIFs), congeners of the clinical candidate rifalazil, to minimize human pregnane X receptor (hPXR) activation while improving potency against MTB. We have determined the compounds' activation of the hPXR [responsible for inducing Cyp450 3A4 (CYP3A4)]. Compound IC50s have been determined against the wild-type and the most prevalent RIFR (ß-S450L) mutant MTB RNAPs. We have also determined their bactericidal activity against "normal" replicating MTB and a model for non-replicating, persister MTB. We have identified a minimal substitution and have probed larger substitutions that exhibit negligible hPXR activation (1.2-fold over the dimethyl sulfoxide control), many of which are 5- to 10-fold more potent against RNAPs and MTB than RMP. Importantly, we have analogues that are essentially equipotent against replicating MTB and non-replicating persister MTB, a property that is correlated with faster kill rates and may lead to shorter treatment durations. This work provides a proof of principle that the ansamycin core remains an attractive and effective scaffold for novel and dramatically improved RIFs.
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Infecções por HIV , Rifamicinas , Tuberculose , Adulto , Infecções por HIV/tratamento farmacológico , Humanos , Receptor de Pregnano X , Rifampina/farmacologia , Rifampina/uso terapêutico , Rifamicinas/farmacologia , Rifamicinas/uso terapêutico , Tuberculose/tratamento farmacológicoRESUMO
Rifampin (RMP), a very potent inhibitor of the Mycobacterium tuberculosis (MTB) RNA polymerase (RNAP), remains a keystone in the treatment of tuberculosis since its introduction in 1965. However, rifamycins suffer from serious drawbacks, including 3- to 9-month treatment times, Cyp450 induction (particularly problematic for HIV-MTB coinfection), and resistant mutations within RNAP that yield RIF-resistant (RIFR) MTB strains. There is a clear and pressing need for improved TB therapies. We have utilized a structure-based drug design approach to synthesize and test novel benzoxazinorifamycins (bxRIF), congeners of the clinical candidate rifalazil. Our goal is to gain binding interactions that will compensate for the loss of RIF-binding affinity to the (RIFR) MTB RNAP and couple those with substitutions that we have previously found that essentially eliminate Cyp450 induction. Herein, we report a systematic exploration of 42 substituted bxRIFs that have yielded an analogue (27a) that has an excellent in vitro activity (MTB RNAP inhibition, MIC, MBC), enhanced (â¼30-fold > RMP) activity against RIFR MTB RNAP, negligible hPXR activation, good mouse pharmacokinetics, and excellent activity with no observable adverse effects in an acute mouse TB model. In a time-kill study, 27a has a 7 day MBC that is â¼10-fold more potent than RMP. These results suggest that 27a may exhibit a faster kill rate than RMP, which could possibly reduce the clinical treatment time. Our synthetic protocol enabled the synthesis of â¼2 g of 27a at >95% purity in 3 months, demonstrating the feasibility of scale-up synthesis of bxRIFs for preclinical and clinical studies.
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Mycobacterium tuberculosis , Rifamicinas , Tuberculose , Animais , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Farmacorresistência Bacteriana , Camundongos , Rifampina/farmacologia , Rifamicinas/farmacologia , Tuberculose/tratamento farmacológicoRESUMO
SARS-CoV-2 proteases Mpro and PLpro are promising targets for antiviral drug development. In this study, we present an antiviral screening strategy involving a novel in-cell protease assay, antiviral and biochemical activity assessments, as well as structural determinations for rapid identification of protease inhibitors with low cytotoxicity. We identified eight compounds with anti-SARS-CoV-2 activity from a library of 64 repurposed drugs and modeled at protease active sites by in silico docking. We demonstrate that Sitagliptin and Daclatasvir inhibit PLpro, and MG-101, Lycorine HCl, and Nelfinavir mesylate inhibit Mpro of SARS-CoV-2. The X-ray crystal structure of Mpro in complex with MG-101 shows a covalent bond formation between the inhibitor and the active site Cys145 residue indicating its mechanism of inhibition is by blocking the substrate binding at the active site. Thus, we provide methods for rapid and effective screening and development of inhibitors for blocking virus polyprotein processing as SARS-CoV-2 antivirals. Additionally, we show that the combined inhibition of Mpro and PLpro is more effective in inhibiting SARS-CoV-2 and the delta variant.
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Proteases 3C de Coronavírus/antagonistas & inibidores , Proteases Semelhantes à Papaína de Coronavírus/antagonistas & inibidores , Avaliação Pré-Clínica de Medicamentos/métodos , SARS-CoV-2/enzimologia , Inibidores de Protease Viral/análise , Reposicionamento de Medicamentos , Células HEK293 , Humanos , Simulação de Acoplamento Molecular , Terapia de Alvo Molecular , Tratamento Farmacológico da COVID-19RESUMO
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.
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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
RNA polymerase (RNAP) is the central enzyme of gene expression, which transcribes DNA to RNA. All cellular organisms synthesize RNA with highly conserved multi-subunit DNA-dependent RNAPs, except mitochondrial RNA transcription, which is carried out by a single-subunit RNAP. Over 60 years of extensive research has elucidated the structures and functions of cellular RNAPs. In this review, we introduce a brief structural feature of bacterial RNAP, the most well characterized model enzyme, and a novel experimental approach known as "Time-dependent soak-trigger-freeze X-ray crystallography" which can be used to observe the RNA synthesis reaction at atomic resolution in real time. This principle methodology can be used for elucidating fundamental mechanisms of cellular RNAP transcription.
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RNA Polimerases Dirigidas por DNA , Transcrição Gênica , Cristalografia por Raios X , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , RNA Bacteriano/genéticaRESUMO
RNA polymerases (RNAPs) synthesize RNA from NTPs, whereas DNA polymerases synthesize DNA from 2'dNTPs. DNA polymerases select against NTPs by using steric gates to exclude the 2'OH, but RNAPs have to employ alternative selection strategies. In single-subunit RNAPs, a conserved Tyr residue discriminates against 2'dNTPs, whereas selectivity mechanisms of multi-subunit RNAPs remain hitherto unknown. Here, we show that a conserved Arg residue uses a two-pronged strategy to select against 2'dNTPs in multi-subunit RNAPs. The conserved Arg interacts with the 2'OH group to promote NTP binding, but selectively inhibits incorporation of 2'dNTPs by interacting with their 3'OH group to favor the catalytically-inert 2'-endo conformation of the deoxyribose moiety. This deformative action is an elegant example of an active selection against a substrate that is a substructure of the correct substrate. Our findings provide important insights into the evolutionary origins of biopolymers and the design of selective inhibitors of viral RNAPs.
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Proteínas de Bactérias/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Desoxirribonucleotídeos/metabolismo , Desoxirribose/metabolismo , Arginina/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/ultraestrutura , Cristalografia por Raios X , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/isolamento & purificação , RNA Polimerases Dirigidas por DNA/ultraestrutura , Escherichia coli/enzimologia , Escherichia coli/genética , Cinética , Simulação de Acoplamento Molecular , Regiões Promotoras Genéticas , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Especificidade por Substrato , Thermus thermophilus/enzimologia , Thermus thermophilus/genéticaRESUMO
Ribosomal RNA (rRNA) is most highly expressed in rapidly growing bacteria and is drastically downregulated under stress conditions by the global transcriptional regulator DksA and the alarmone ppGpp. Here, we determined cryo-electron microscopy structures of the Escherichia coli RNA polymerase (RNAP) σ70 holoenzyme during rRNA promoter recognition with and without DksA/ppGpp. RNAP contacts the UP element using dimerized α subunit carboxyl-terminal domains and scrunches the template DNA with the σ finger and ß' lid to select the transcription start site favorable for rapid promoter escape. Promoter binding induces conformational change of σ domain 2 that opens a gate for DNA loading and ejects σ1.1 from the RNAP cleft to facilitate open complex formation. DksA/ppGpp binding also opens the DNA loading gate, which is not coupled to σ1.1 ejection and impedes open complex formation. These results provide a molecular basis for the exceptionally active rRNA transcription and its vulnerability to DksA/ppGpp.
Assuntos
Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Regiões Promotoras Genéticas/genética , RNA Ribossômico/genética , Transcrição Gênica , Microscopia Crioeletrônica , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/metabolismo , RNA Polimerases Dirigidas por DNA/ultraestrutura , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Guanosina Tetrafosfato/metabolismo , Holoenzimas/química , Holoenzimas/metabolismo , Holoenzimas/ultraestrutura , Conformação Proteica , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA Ribossômico/metabolismo , Fator sigma/química , Fator sigma/metabolismo , Fator sigma/ultraestrutura , Sítio de Iniciação de TranscriçãoRESUMO
Opening of the DNA binding cleft of cellular RNA polymerase (RNAP) is necessary for transcription initiation but the underlying molecular mechanism is not known. Here, we report on the cryo-electron microscopy structures of the RNAP, RNAP-TFEα binary, and RNAP-TFEα-promoter DNA ternary complexes from archaea, Thermococcus kodakarensis (Tko). The structures reveal that TFEα bridges the RNAP clamp and stalk domains to open the DNA binding cleft. Positioning of promoter DNA into the cleft closes it while maintaining the TFEα interactions with the RNAP mobile modules. The structures and photo-crosslinking results also suggest that the conserved aromatic residue in the extended winged-helix domain of TFEα interacts with promoter DNA to stabilize the transcription bubble. This study provides a structural basis for the functions of TFEα and elucidates the mechanism by which the DNA binding cleft is opened during transcription initiation in the stalk-containing RNAPs, including archaeal and eukaryotic RNAPs.