RESUMO
T-box riboswitches are noncoding RNA elements involved in genetic regulation of most Gram-positive bacteria. They regulate amino acid metabolism by assessing the aminoacylation status of tRNA, subsequently affecting the transcription or translation of downstream amino acid metabolism-related genes. Here we present single-molecule FRET studies of the Mycobacterium tuberculosis IleS T-box riboswitch, a paradigmatic translational T-box. Results support a two-step binding model, where the tRNA anticodon is recognized first, followed by interactions with the NCCA sequence. Furthermore, after anticodon recognition, tRNA can transiently dock into the discriminator domain even in the absence of the tRNA NCCA-discriminator interactions. Establishment of the NCCA-discriminator interactions significantly stabilizes the fully bound state. Collectively, the data suggest high conformational flexibility in translational T-box riboswitches; and supports a conformational selection model for NCCA recognition. These findings provide a kinetic framework to understand how specific RNA elements underpin the binding affinity and specificity required for gene regulation.
Assuntos
Anticódon , Mycobacterium tuberculosis , Conformação de Ácido Nucleico , RNA Bacteriano , RNA de Transferência , Riboswitch , Riboswitch/genética , RNA de Transferência/metabolismo , RNA de Transferência/genética , RNA de Transferência/química , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/genética , Anticódon/metabolismo , Anticódon/genética , RNA Bacteriano/metabolismo , RNA Bacteriano/genética , RNA Bacteriano/química , Transferência Ressonante de Energia de Fluorescência , Biossíntese de Proteínas , Regulação Bacteriana da Expressão Gênica , CinéticaRESUMO
Topoisomerase I (TOP1) is an essential enzyme that relaxes DNA to prevent and dissipate torsional stress during transcription. However, the mechanisms underlying the regulation of TOP1 activity remain elusive. Using enhanced cross-linking and immunoprecipitation (eCLIP) and ultraviolet-cross-linked RNA immunoprecipitation followed by total RNA sequencing (UV-RIP-seq) in human colon cancer cells along with RNA electrophoretic mobility shift assays (EMSAs), biolayer interferometry (BLI), and in vitro RNA-binding assays, we identify TOP1 as an RNA-binding protein (RBP). We show that TOP1 directly binds RNA in vitro and in cells and that most RNAs bound by TOP1 are mRNAs. Using a TOP1 RNA-binding mutant and topoisomerase cleavage complex sequencing (TOP1cc-seq) to map TOP1 catalytic activity, we reveal that RNA opposes TOP1 activity as RNA polymerase II (RNAPII) commences transcription of active genes. We further demonstrate the inhibitory role of RNA in regulating TOP1 activity by employing DNA supercoiling assays and magnetic tweezers. These findings provide insight into the coordinated actions of RNA and TOP1 in regulating DNA topological stress intrinsic to RNAPII-dependent transcription.
RESUMO
Streptococcus pneumoniae (the pneumococcus) is the major cause of bacterial pneumonia in the US and worldwide. Studies have shown that the differing chemical make-up between serotypes of its most important virulence factor, the capsule, can dictate disease severity. Here we demonstrate that control of capsule synthesis is also critical for infection and facilitated by two broadly conserved transcription factors, SpxR and CpsR, through a distal cis-regulatory element we name the 37-CE. Strikingly, changing only three nucleotides within this sequence is sufficient to render pneumococcus avirulent. Using in vivo and in vitro approaches, we present a model where SpxR interacts as a unique trimeric quaternary structure with the 37-CE to enable capsule repression in the airways. Considering its dramatic effect on infection, variation of the 37-CE between serotypes suggests this molecular switch could be a critical contributing factor to this pathogen's serotype-specific disease outcomes.
Assuntos
Infecções Pneumocócicas , Streptococcus pneumoniae , Humanos , Streptococcus pneumoniae/metabolismo , Infecções Pneumocócicas/microbiologia , Fatores de Virulência/metabolismo , Sistema Respiratório/metabolismo , Sequências Reguladoras de Ácido Nucleico , Sorogrupo , Cápsulas Bacterianas/genética , Cápsulas Bacterianas/metabolismoRESUMO
Topoisomerase V is a unique topoisomerase that combines DNA repair and topoisomerase activities. The enzyme has an unusual arrangement, with a small topoisomerase domain followed by 12 tandem (HhH)2 domains, which include 3 AP lyase repair domains. The uncommon architecture of this enzyme bears no resemblance to any other known topoisomerase. Here, we present structures of topoisomerase V in complex with DNA. The structures show that the (HhH)2 domains wrap around the DNA and in this manner appear to act as a processivity factor. There is a conformational change in the protein to expose the topoisomerase active site. The DNA bends sharply to enter the active site, which melts the DNA and probably facilitates relaxation. The structures show a DNA-binding mode not observed before and provide information on the way this atypical topoisomerase relaxes DNA. In common with type IB enzymes, topoisomerase V relaxes DNA using a controlled rotation mechanism, but the structures show that topoisomerase V accomplishes this in different manner. Overall, the structures firmly establish that type IC topoisomerases form a distinct type of topoisomerases, with no similarities to other types at the sequence, structural, or mechanistic level. They represent a completely different solution to DNA relaxation.
Assuntos
DNA Liase (Sítios Apurínicos ou Apirimidínicos) , Euryarchaeota , DNA/metabolismo , DNA Topoisomerases Tipo I/genética , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/química , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Euryarchaeota/genética , Euryarchaeota/metabolismoRESUMO
Clustered regularly interspaced short palindromic repeats (CRISPR) and their CRISPR-associated proteins (Cas) provide many prokaryotes with an adaptive immune system against invading genetic material. Type III CRISPR systems are unique in that they can degrade both RNA and DNA. In response to invading nucleic acids, they produce cyclic oligoadenylates that act as secondary messengers, activating cellular nucleases that aid in the immune response. Here, we present seven single-particle cryo-EM structures of the type III-A Staphylococcus epidermidis CRISPR effector complex. The structures reveal the intact S. epidermidis effector complex in an apo, ATP-bound, cognate target RNA-bound, and non-cognate target RNA-bound states and illustrate how the effector complex binds and presents crRNA. The complexes bound to target RNA capture the type III-A effector complex in a post-RNA cleavage state. The ATP-bound structures give details about how ATP binds to Cas10 to facilitate cyclic oligoadenylate production.
Assuntos
Proteínas Associadas a CRISPR , Trifosfato de Adenosina/metabolismo , Proteínas Associadas a CRISPR/química , Proteínas Associadas a CRISPR/genética , Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , RNA/metabolismo , Staphylococcus epidermidis/genética , Staphylococcus epidermidis/metabolismoRESUMO
Ribozymes are folded catalytic RNA molecules that perform important biological functions. Since the discovery of the first RNA with catalytic activity in 1982, a large number of ribozymes have been reported. While most catalytic RNA molecules act alone, some RNA-based catalysts, such as RNase P, the ribosome, and the spliceosome, need protein components to perform their functions in the cell. In the last decades, the structure and mechanism of several ribozymes have been studied in detail. Aside from the ribosome, which catalyzes peptide bond formation during protein synthesis, the majority of known ribozymes carry out mostly phosphoryl transfer reactions, notably trans-esterification or hydrolysis reactions. In this review, we describe the main features of the mechanisms of various types of ribozymes that can function with or without the help of proteins to perform their biological functions.
Assuntos
RNA Catalítico/metabolismo , Catálise , Ribossomos/metabolismo , Spliceossomos/metabolismoRESUMO
DNA gyrase is a type II topoisomerase that is responsible for maintaining the topological state of bacterial and some archaeal genomes. It uses an ATP-dependent two-gate strand-passage mechanism that is shared among all type II topoisomerases. During this process, DNA gyrase creates a transient break in the DNA, the G-segment, to form a cleavage complex. This allows a second DNA duplex, known as the T-segment, to pass through the broken G-segment. After the broken strand is religated, the T-segment is able to exit out of the enzyme through a gate called the C-gate. Although many steps of the type II topoisomerase mechanism have been studied extensively, many questions remain about how the T-segment ultimately exits out of the C-gate. A recent cryo-EM structure of Streptococcus pneumoniae GyrA shows a putative T-segment in close proximity to the C-gate, suggesting that residues in this region may be important for coordinating DNA exit from the enzyme. Here, we show through site-directed mutagenesis and biochemical characterization that three conserved basic residues in the C-gate of DNA gyrase are important for DNA supercoiling activity, but not for ATPase or cleavage activity. Together with the structural information previously published, our data suggest a model in which these residues cluster to form a positively charged region that facilitates T-segment passage into the cavity formed between the DNA gate and C-gate.
Assuntos
Domínio Catalítico , DNA Girase/metabolismo , DNA Bacteriano/química , DNA Super-Helicoidal , Infecções Pneumocócicas/enzimologia , Elementos Estruturais de Proteínas , Streptococcus pneumoniae/enzimologia , DNA Girase/química , DNA Topoisomerases Tipo II/química , DNA Topoisomerases Tipo II/metabolismo , Modelos Moleculares , Mutagênese Sítio-Dirigida/métodos , Infecções Pneumocócicas/microbiologia , Infecções Pneumocócicas/patologia , Streptococcus pneumoniae/isolamento & purificação , Streptococcus pneumoniae/patogenicidadeRESUMO
Cobalamin riboswitches encompass a structurally diverse group of cis-acting, gene regulatory elements found mostly in bacterial messenger RNA and are classified into subtypes based on secondary and tertiary characteristics. An unusual variant of the cobalamin riboswitch with predicted structural features was identified in Bacillus subtilis over a decade ago, but its structure and mechanisms of cobalamin selectivity and translational control have remained unsolved. We present the crystal structure of the aptamer domain of this atypical cobalamin riboswitch and a model for the complete riboswitch, including its expression platform domain. We demonstrate that this riboswitch binds to multiple cobalamin derivatives and correlate its promiscuous behavior to its structure and unique arrangement of peripheral elements. Comparative structural analyses between conventional cobalamin riboswitches and the B. subtilis cobalamin riboswitch reveal that the likely basis for this promiscuous ligand binding is intrinsic structural adaptability encoded in the RNA structure. It suggests that cobalamin selectivity might ultimately be viewed as existing on a spectrum of affinity for each derivative rather than as belonging to distinct types based on ligand specificities. Our work provides an interesting and notable example of functional coupling of ligand-sensing and adaptive folding by a structured RNA molecule.
Assuntos
Aptâmeros de Nucleotídeos/química , Cobamidas/química , Dobramento de RNA , Riboswitch , Vitamina B 12/química , Bacillus subtilisRESUMO
Ubiquitous across all domains of life, tRNAs constitute an essential component of cellular physiology, carry out an indispensable role in protein synthesis, and have been historically the subject of a wide range of biochemical and biophysical studies as prototypical folded RNA molecules. Although conformational flexibility is a well-established characteristic of tRNA structure, it is typically regarded as an adaptive property exhibited in response to an inducing event, such as the binding of a tRNA synthetase or the accommodation of an aminoacyl-tRNA into the ribosome. In this study, we present crystallographic data of a tRNA molecule to expand on this paradigm by showing that structural flexibility and plasticity are intrinsic properties of tRNAs, apparent even in the absence of other factors. Based on two closely related conformations observed within the same crystal, we posit that unbound tRNAs by themselves are flexible and dynamic molecules. Furthermore, we demonstrate that the formation of the T-loop conformation by the tRNA TΨC stem-loop, a well-characterized and classic RNA structural motif, is possible even in the absence of important interactions observed in fully folded tRNAs.
Assuntos
Conformação de Ácido Nucleico , Aminoacil-RNA de Transferência/ultraestrutura , RNA de Transferência/ultraestrutura , Anticódon/química , Anticódon/genética , Cristalografia , Escherichia coli/química , Escherichia coli/ultraestrutura , Motivos de Nucleotídeos/genética , RNA de Transferência/química , Aminoacil-RNA de Transferência/química , Ribossomos/genética , Ribossomos/ultraestruturaRESUMO
Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated Cas proteins provide an immune-like response in many prokaryotes against extraneous nucleic acids. CRISPR-Cas systems are classified into different classes and types. Class 1 CRISPR-Cas systems form multi-protein effector complexes that includes a guide RNA (crRNA) used to identify the target for destruction. Here we present crystal structures of Staphylococcus epidermidis Type III-A CRISPR subunits Csm2 and Csm3 and a 5.2 Å resolution single-particle cryo-electron microscopy (cryo-EM) reconstruction of an in vivo assembled effector subcomplex including the crRNA. The structures help to clarify the quaternary architecture of Type III-A effector complexes, and provide details on crRNA binding, target RNA binding and cleavage, and intermolecular interactions essential for effector complex assembly. The structures allow a better understanding of the organization of Type III-A CRISPR effector complexes as well as highlighting the overall similarities and differences with other Class 1 effector complexes.
Assuntos
Proteínas Associadas a CRISPR/ultraestrutura , Conformação Proteica , Staphylococcus epidermidis/química , Proteínas Associadas a CRISPR/química , Sistemas CRISPR-Cas/genética , Microscopia Crioeletrônica , Cristalografia por Raios X , RNA Bacteriano/química , RNA Guia de Cinetoplastídeos/química , RNA Guia de Cinetoplastídeos/genética , Staphylococcus epidermidis/genéticaRESUMO
Gyrase is a unique type IIA topoisomerase that uses ATP hydrolysis to maintain the negatively supercoiled state of bacterial DNA. In order to perform its function, gyrase undergoes a sequence of conformational changes that consist of concerted gate openings, DNA cleavage, and DNA strand passage events. Structures where the transported DNA molecule (T-segment) is trapped by the A subunit have not been observed. Here we present the cryoEM structures of two oligomeric complexes of open gyrase A dimers and DNA. The protein subunits in these complexes were solved to 4 Å and 5.2 Å resolution. One of the complexes traps a linear DNA molecule, a putative T-segment, which interacts with the open gyrase A dimers in two states, representing steps either prior to or after passage through the DNA-gate. The structures locate the T-segment in important intermediate conformations of the catalytic cycle and provide insights into gyrase-DNA interactions and mechanism.
Assuntos
Trifosfato de Adenosina/química , DNA Girase/química , DNA Bacteriano/química , Escherichia coli/genética , Subunidades Proteicas/química , Streptococcus pneumoniae/genética , Trifosfato de Adenosina/metabolismo , Sítios de Ligação , Clonagem Molecular , Microscopia Crioeletrônica , Clivagem do DNA , DNA Girase/metabolismo , DNA Bacteriano/metabolismo , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Isoenzimas/química , Isoenzimas/metabolismo , Modelos Moleculares , Oligonucleotídeos/química , Oligonucleotídeos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Subunidades Proteicas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Streptococcus pneumoniae/metabolismoRESUMO
T-box riboswitches are cis-regulatory RNA elements that regulate the expression of proteins involved in amino acid biosynthesis and transport by binding to specific tRNAs and sensing their aminoacylation state. While the T-box modular structural elements that recognize different parts of a tRNA have been identified, the kinetic trajectory describing how these interactions are established temporally remains unclear. Using smFRET, we demonstrate that tRNA binds to the riboswitch in two steps, first anticodon recognition followed by the sensing of the 3' NCCA end, with the second step accompanied by a T-box riboswitch conformational change. Studies on site-specific mutants highlight that specific T-box structural elements drive the two-step binding process in a modular fashion. Our results set up a kinetic framework describing tRNA binding by T-box riboswitches, and suggest such binding mechanism is kinetically beneficial for efficient, co-transcriptional recognition of the cognate tRNA ligand.
Assuntos
Anticódon/genética , Genes Reguladores/genética , RNA de Transferência/genética , Riboswitch/genética , Aminoácidos/genética , Bacillus subtilis/genética , Sítios de Ligação/genética , Cinética , Ligantes , Conformação de Ácido NucleicoRESUMO
Ribonuclease P (RNase P) catalyzes the removal of 5' leaders of tRNA precursors and its central catalytic RNA subunit is highly conserved across all domains of life. In eukaryotes, RNase P and RNase MRP, a closely related ribonucleoprotein enzyme, share several of the same protein subunits, contain a similar catalytic RNA core, and exhibit structural features that do not exist in their bacterial or archaeal counterparts. A unique feature of eukaryotic RNase P/MRP is the presence of two relatively long and unpaired internal loops within the P3 region of their RNA subunit bound by a heterodimeric protein complex, Rpp20/Rpp25. Here we present a crystal structure of the human Rpp20/Rpp25 heterodimer and we propose, using comparative structural analyses, that the evolutionary divergence of the single-stranded and helical nucleic acid binding specificities of eukaryotic Rpp20/Rpp25 and their related archaeal Alba chromatin protein dimers, respectively, originate primarily from quaternary level differences observed in their heterodimerization interface. Our work provides structural insights into how the archaeal Alba protein scaffold was adapted evolutionarily for incorporation into several functionally-independent eukaryotic ribonucleoprotein complexes.
Assuntos
Autoantígenos/química , Autoantígenos/metabolismo , RNA/metabolismo , Ribonuclease P/química , Ribonuclease P/metabolismo , Proteínas Arqueais/química , Proteínas Arqueais/metabolismo , Cristalografia por Raios X , Evolução Molecular , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Humanos , Modelos Moleculares , Complexos Multienzimáticos/química , Complexos Multienzimáticos/metabolismo , Domínios Proteicos , Multimerização Proteica , Estrutura Quaternária de Proteína , Estrutura Secundária de ProteínaRESUMO
Magnetic tweezers (MT) provide a powerful single-molecule approach to study the mechanism of topoisomerases, giving the experimenter the ability to change and read out DNA topology in real time. By using diverse DNA substrates, one can study different aspects of topoisomerase function and arrive at a better mechanistic understanding of these fascinating enzymes. Here we describe methods for the creation of three different DNA substrates used in MT experiments with topoisomerases: double-stranded DNA (dsDNA) tethers, "braided" (intertwined or catenated) DNA tether pairs, and dsDNA tethers with single-stranded DNA (ssDNA) regions. Additionally, we discuss how to build flow cells for bright-field MT microscopy, as well as how to noncovalently attach anti-digoxigenin to the coverslip surface for tethering digoxigenin-labeled DNAs. Finally, we describe procedures for the identification of a suitable DNA substrate for MT study and data collection.
Assuntos
DNA Topoisomerases Tipo I/metabolismo , Imagem Individual de Molécula/instrumentação , DNA/metabolismo , DNA de Cadeia Simples/metabolismo , Conformação de Ácido NucleicoRESUMO
We study the statistical-mechanical properties of intertwined double-helical DNAs (DNA braids). In magnetic tweezers experiments, we find that torsionally stressed stretched braids supercoil via an abrupt buckling transition, which is associated with the nucleation of a braid end loop, and that the buckled braid is characterized by a proliferation of multiple domains. Differences between the mechanics of DNA braids and supercoiled single DNAs can be understood as an effect of the increased bulkiness in the structure of the former. The experimental results are in accord with the predictions of a statistical-mechanical model.
Assuntos
DNA Super-Helicoidal/química , Conformação de Ácido Nucleico , DNA/química , MagnetismoRESUMO
A T-box regulator or riboswitch actively monitors the levels of charged/uncharged tRNA and participates in amino acid homeostasis by regulating genes involved in their utilization or biosynthesis. It has an aptamer domain for cognate tRNA recognition and an expression platform to sense the charge state and modulate gene expression. These two conserved domains are connected by a variable linker that harbors additional secondary structural elements, such as Stem III. The structural basis for specific tRNA binding is known, but the structural basis for charge sensing and the role of other elements remains elusive. To gain new structural insights on the T-box mechanism, a molecular envelope was calculated from small angle X-ray scattering data for the Bacillus subtilis glyQS T-box riboswitch in complex with an uncharged tRNAGly. A structural model of an anti-terminated glyQS T-box in complex with its cognate tRNAGly was derived based on the molecular envelope. It shows the location and relative orientation of various secondary structural elements. The model was validated by comparing the envelopes of the wild-type complex and two variants. The structural model suggests that in addition to a possible regulatory role, Stem III could aid in preferential stabilization of the T-box anti-terminated state allowing read-through of regulated genes.
Assuntos
RNA Bacteriano/química , RNA Bacteriano/metabolismo , RNA de Transferência de Glicina/química , RNA de Transferência de Glicina/metabolismo , Riboswitch/genética , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Sequência de Bases , Modelos Moleculares , Conformação de Ácido Nucleico , RNA Bacteriano/genética , RNA de Transferência de Glicina/genética , Espalhamento a Baixo Ângulo , Difração de Raios XRESUMO
Topoisomerases are enzymes that are involved in maintaining the topological state of cellular DNA. Their dynamic characteristics remain poorly understood despite numerous structural, biophysical and biochemical studies. Recent single-molecule experiments revealed that an important feature of the type IA topoisomerase mechanism is the presence of pauses between relaxation events. However, these experiments could not determine whether the protein remains DNA bound during the pauses or what relationship may exist between protein domain movements and topological changes in the DNA. By combining two orthogonal single-molecule techniques, we found that E. coli topoisomerase I constantly changes conformation when attempting to modify the topology of DNA, but succeeds in only a fraction of the attempts. Thus, its mechanism can be described as a series of DNA strand-passage attempts that culminate in a successful relaxation event.
Assuntos
DNA Topoisomerases Tipo I/química , DNA Topoisomerases Tipo I/metabolismo , DNA/metabolismo , Escherichia coli/enzimologia , Modelos Biológicos , Modelos Moleculares , Conformação de Ácido Nucleico , Conformação ProteicaRESUMO
Topoisomerase V (Topo-V) is the only topoisomerase with both topoisomerase and DNA repair activities. The topoisomerase activity is conferred by a small alpha-helical domain, whereas the AP lyase activity is found in a region formed by 12 tandem helix-hairpin-helix ((HhH)2) domains. Although it was known that Topo-V has multiple repair sites, only one had been mapped. Here, we show that Topo-V has three AP lyase sites. The atomic structure and Small Angle X-ray Scattering studies of a 97 kDa fragment spanning the topoisomerase and 10 (HhH)2 domains reveal that the (HhH)2 domains extend away from the topoisomerase domain. A combination of biochemical and structural observations allow the mapping of the second repair site to the junction of the 9th and 10th (HhH)2 domains. The second site is structurally similar to the first one and to the sites found in other AP lyases. The 3rd AP lyase site is located in the 12th (HhH)2 domain. The results show that Topo-V is an unusual protein: it is the only known protein with more than one (HhH)2 domain, the only known topoisomerase with dual activities and is also unique by having three AP lyase repair sites in the same polypeptide.
Assuntos
DNA Topoisomerases Tipo I/química , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/química , Euryarchaeota/enzimologia , Domínio Catalítico , Modelos Moleculares , Estrutura Terciária de ProteínaRESUMO
Type II CRISPR systems defend against invasive DNA by using Cas9 as an RNA-guided nuclease that creates double-stranded DNA breaks. Dual RNAs (CRISPR RNA [crRNA] and tracrRNA) are required for Cas9's targeting activities observed to date. Targeting requires a protospacer adjacent motif (PAM) and crRNA-DNA complementarity. Cas9 orthologs (including Neisseria meningitidis Cas9 [NmeCas9]) have also been adopted for genome engineering. Here we examine the DNA cleavage activities and substrate requirements of NmeCas9, including a set of unusually complex PAM recognition patterns. Unexpectedly, NmeCas9 cleaves single-stranded DNAs in a manner that is RNA guided but PAM and tracrRNA independent. Beyond the need for guide-target pairing, this "DNase H" activity has no apparent sequence requirements, and the cleavage sites are measured from the 5' end of the DNA substrate's RNA-paired region. These results indicate that tracrRNA is not strictly required for NmeCas9 enzymatic activation, and expand the list of targeting activities of Cas9 endonucleases.
Assuntos
Proteínas de Bactérias/genética , Sistemas CRISPR-Cas , DNA Bacteriano/genética , Endodesoxirribonucleases/genética , Neisseria meningitidis/genética , Proteínas de Bactérias/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , DNA Bacteriano/metabolismo , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Endodesoxirribonucleases/metabolismo , Dados de Sequência Molecular , Neisseria meningitidis/enzimologia , Motivos de Nucleotídeos , RNA Guia de Cinetoplastídeos/genética , RNA Guia de Cinetoplastídeos/metabolismoRESUMO
Many transcriptional activators act at a distance from core promoter elements and work by recruiting RNA polymerase through protein-protein interactions. We show here how the prokaryotic regulatory protein CueR both represses and activates transcription by differentially modulating local DNA structure within the promoter. Structural studies reveal that the repressor state slightly bends the promoter DNA, precluding optimal RNA polymerase-promoter recognition. Upon binding a metal ion in the allosteric site, CueR switches into an activator conformation. It maintains all protein-DNA contacts but introduces torsional stresses that kink and undertwist the promoter, stabilizing an A-form DNA-like conformation. These factors switch on and off transcription by exerting dynamic control of DNA stereochemistry, reshaping the core promoter and making it a better or worse substrate for polymerase.