RESUMO
XACT-seq ("crosslink between active-center and template sequencing") is a technique for high-throughput, single-nucleotide resolution mapping of RNA polymerase (RNAP) active-center positions relative to the DNA template. XACT-seq overcomes limitations of approaches that rely on analysis of the RNA 3' end (e.g., native elongating transcript sequencing) or that report RNAP positions with low resolution (e.g., ChIP-seq and ChIP-exo). XACT-seq can be used to map RNAP active-center positions in transcription initiation complexes, initially transcribing complexes, and transcription elongation complexes. For complete details on the use and execution of this protocol, please refer to Winkelman et al. (2020).
Assuntos
DNA Bacteriano/genética , RNA Polimerases Dirigidas por DNA/genética , Escherichia coli/genética , Técnicas Genéticas , Ensaios de Triagem em Larga Escala/métodos , RNA Polimerases Dirigidas por DNA/efeitos da radiação , Transcrição Gênica/genética , Raios UltravioletaRESUMO
We report the mutational spectra in a segment of the E. coli rpoB gene of bleomycin (BLEO), 4-nitroquinoline-1-oxide (NQO), and hydrogen peroxide (H2O2). We compare these spectra with those of other mutagens and repair deficient strains in the same rpoB system, and review the key elements determining mutational hotspots and outline the questions that remain unanswered. We consider three tiers of hotspots that derive from 1) the nature of the sequence change at a specific base, 2) the direct nearest neighbors and 3) some aspect of the larger sequence context or the local 3D-structure of segments of DNA. This latter tier can have a profound effect on mutation frequencies, even among sites with identical nearest neighbor sequences. BLEO is dependent on the SOS-induced translesion Pol V for mutagenesis, and has a dramatic hotspot at a single mutational site in rpoB. NQO is not dependent on any of the translesion polymerases, in contrast to findings with plasmids treated in vitro and transformed into E. coli. The rpoB system allows one to monitor both G:C -> A:T transitions and G:C -> T:A transversions at the same site in 11 cases, each site having the identical sequence context for each of the two mutations. The combined preference for G:C -> A:T transitions at these sites is 20-fold. Several of the favored sites for hydrogen peroxide mutagenesis are not seen in the spectra of BLEO and NQO mutations, indicating that mutagenesis from reactive oxygen species is not a major cause of BLEO or NQO mutagenesis, but rather specific adducts. The variance in mutation rates at sites with identical nearest neighbors suggests that the local structure of different DNA segments is an important factor in mutational hotspots.
Assuntos
4-Nitroquinolina-1-Óxido/toxicidade , Bleomicina/toxicidade , RNA Polimerases Dirigidas por DNA/genética , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/efeitos da radiação , Peróxido de Hidrogênio/toxicidade , Mutação , Antibióticos Antineoplásicos/toxicidade , RNA Polimerases Dirigidas por DNA/efeitos da radiação , Escherichia coli/efeitos da radiação , Proteínas de Escherichia coli/efeitos da radiação , Mutagênicos/toxicidade , Oxidantes/toxicidadeAssuntos
DNA/genética , Luz , Hibridização de Ácido Nucleico/efeitos da radiação , Ácidos Nucleicos Peptídicos/genética , Compostos Azo , RNA Polimerases Dirigidas por DNA/efeitos da radiação , Oligodesoxirribonucleotídeos , Transcrição Gênica/efeitos da radiação , Proteínas Virais/efeitos da radiaçãoRESUMO
Thermal variations can exert dramatic effects on the rates of enzymes. The influence of temperature on RNA polymerase is of particular interest because its transcriptional activity governs general levels of gene expression, and may therefore exhibit pleiotropic effects in cells. Using a custom-modified optical trapping apparatus, we used a tightly focused infrared laser to heat single molecules of Escherichia coli RNA polymerase while monitoring transcriptional activity. We found a significant change in rates of transcript elongation with temperature, consistent with a large enthalpic barrier to the condensation reaction associated with RNA polymerization (approximately 13 kcal/mol). In contrast, we found little change in either the frequency or the lifetime of off-pathway, paused states, indicating that the energetic barrier to transcriptional pausing is predominantly entropic.
Assuntos
Calorimetria/métodos , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/enzimologia , Nanotecnologia/métodos , Transcrição Gênica/fisiologia , Ativação Transcricional/fisiologia , RNA Polimerases Dirigidas por DNA/efeitos da radiação , Ativação Enzimática/efeitos da radiação , Escherichia coli/efeitos da radiação , Temperatura Alta , Lasers , Microquímica/métodos , Transcrição Gênica/efeitos dos fármacos , Ativação Transcricional/efeitos dos fármacosRESUMO
Organisms rely on close interplay between DNA replication, recombination, and repair to secure transmission of the genome. In rapidly dividing cells, there is also great pressure for transcription, which may induce conflict with replication. We investigated the potential for conflict in bacterial cells, where there is no temporal separation of these processes. Eliminating the stringent response regulators ppGpp and DksA or the GreA and Mfd proteins, which revive or dislodge stalled transcription complexes, and especially combinations of these factors, is shown to severely reduce viability when DNA repair is also compromised. Both ppGpp and certain RNA polymerase (RNAP) mutations reduce accumulation of backed-up arrays of stalled transcription complexes. We propose these arrays are formidable obstacles to replication that are normally kept in check in wild-type cells by ppGpp, DksA, GreA, and Mfd. When arrays do obstruct replication, the consequences are resolved by one of the many pathways available to rescue stalled forks.
Assuntos
Reparo do DNA/fisiologia , Replicação do DNA/genética , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/fisiologia , Transcrição Gênica/genética , Reparo do DNA/genética , Reparo do DNA/efeitos da radiação , Replicação do DNA/fisiologia , Replicação do DNA/efeitos da radiação , RNA Polimerases Dirigidas por DNA/efeitos da radiação , Escherichia coli K12/crescimento & desenvolvimento , Escherichia coli K12/metabolismo , Escherichia coli K12/efeitos da radiação , Guanosina Tetrafosfato/metabolismo , Mutação , Fatores de Tempo , Transcrição Gênica/fisiologia , Transcrição Gênica/efeitos da radiação , Raios UltravioletaRESUMO
Cyclobutane pyrimidine dimers (CPDs) are directly involved in signaling for UV-induced apoptosis in mammalian cells. Failure to remove these lesions, specially those located at actively expressing genes, is critical, as cells defective in transcription coupled repair have increased apoptotic levels. Thus, the blockage of RNA synthesis by lesions is an important candidate event triggering off active cell death. In this work, wild-type and XPB mutated Chinese hamster ovary (CHO) cells expressing a marsupial photolyase, that removes specifically CPDs from the damaged DNA, were generated, in order to investigate the importance of this lesion in both RNA transcription blockage and apoptotic induction. Photorepair strongly recovers RNA synthesis in wild-type CHO cell line, although the resumption of transcription is decreased in XPB deficient cells. This recovery is accompanied by the prevention of cells entering into apoptosis. These results demonstrate that marsupial photolyase has access to CPDs blocking RNA synthesis in vivo, and this may be affected by the presence of a mutated XPB protein.
Assuntos
Apoptose/fisiologia , Reparo do DNA/fisiologia , Proteínas de Ligação a DNA/deficiência , RNA Polimerases Dirigidas por DNA/metabolismo , Células Eucarióticas/enzimologia , Dímeros de Pirimidina/metabolismo , RNA/biossíntese , Animais , Apoptose/efeitos da radiação , Células CHO , Cricetinae , DNA Helicases , Reparo do DNA/efeitos da radiação , Proteínas de Ligação a DNA/genética , RNA Polimerases Dirigidas por DNA/efeitos da radiação , Desoxirribodipirimidina Fotoliase/genética , Relação Dose-Resposta à Radiação , Células Eucarióticas/efeitos da radiação , Mutação/genética , Dímeros de Pirimidina/antagonistas & inibidores , RNA/genética , Raios UltravioletaRESUMO
Environmental electromagnetic fields have been implicated in human cancers. We examined whether high extremely low frequency (ELF) AC magnetic fields could affect DNA synthesis, transcription or repair, using in vitro model systems with defined sequences. The rate and fidelity of DNA polymerase catalyzed DNA synthesis, as well as of RNA polymerase catalyzed RNA synthesis, were not statistically significantly affected by 60 Hz 0.25-0.5 Tesla magnetic fields. The efficiency of mutS dependent mismatch repair with human cell extracts was also not affected by the magnetic field exposure. The results suggest that the core processes related to the transmission of genetic information are stable under high ELF magnetic fields.
Assuntos
Pareamento Incorreto de Bases , Reparo do DNA , DNA Polimerase Dirigida por DNA/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Campos Eletromagnéticos , Animais , Sistema Livre de Células , DNA/biossíntese , Reparo do DNA/efeitos da radiação , DNA Polimerase Dirigida por DNA/efeitos da radiação , RNA Polimerases Dirigidas por DNA/efeitos da radiação , Escherichia coli/genética , Escherichia coli/efeitos da radiação , Células HeLa , Humanos , Cinética , RNA/biossínteseRESUMO
We have dissected the steps in T7 RNA polymerase transcription initiation using psoralen cross-linking. DNA templates containing cross-links at either -14/-13, -2/-1, or -4/-3 were constructed. These cross-links are within the DNA-contacting region in the initiation complex. A cross-link at -2/-1 did not affect T7 RNA polymerase binding affinity, whereas a cross-link at -14/-13 reduced binding affinity by less than 2-fold. Transcription initiation was completely blocked by cross-links at -14/-13 or at -2/-1. A cross-link at -4/-3 inhibited neither binding nor the first RNA phosphodiester bond but greatly inhibited further RNA chain extension. Circular dichroism spectroscopy revealed that DNA melting in the -4/-3 cross-link was greatly inhibited, indicating that inhibition of RNA chain extension was a melting defect. Transcription shutoff on the -14/-13 cross-link may be due to inhibition of conformational changes in the polymerase-DNA complex. Because the -2/-1 cross-link is immediately upstream of the start site (+1), open complex formation may have been completely inhibited by this cross-link, accounting for the shutoff of transcription. Thus, depending on their location, psoralen cross-links affected different steps in the initiation process. We propose that promoter melting is progressive and that melting of one or two bp upstream of the +1 site is sufficient for formation of the first phosphodiester bond while further RNA chain extension within the promoter depends on greater upstream melting of the promoter, which may be required for stabilization of the initiation complex.
Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , DNA/metabolismo , Metoxaleno/farmacologia , Regiões Promotoras Genéticas , Conformação Proteica , Transcrição Gênica/efeitos dos fármacos , Bacteriófago T7/enzimologia , Sequência de Bases , Dicroísmo Circular , Reagentes de Ligações Cruzadas , DNA/química , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/efeitos da radiação , Cinética , Luz , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Oligodesoxirribonucleotídeos/química , Fotoquímica , Desnaturação Proteica , Moldes Genéticos , Transcrição Gênica/efeitos da radiação , Proteínas ViraisRESUMO
We report that high energy beta particles may function as a means for mapping the surface of a protein. Comparable to Fe-EDTA in the presence of ascorbate and peroxide, 90Y-EDTA alone can break polypeptide backbone bonds on the surface of E. coli RNA polymerase. The two methods give very similar fragmentation patterns, although some unique fragments are produced by each. Radiolytic footprinting may prove useful for mapping proteins inside living cells, since beta-radiolysis produces reactive species up to approximately 1 cm away from the emitting 90Y.
Assuntos
RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/efeitos da radiação , Proteínas/química , Proteínas/efeitos da radiação , Partículas beta , Fenômenos Biofísicos , Biofísica , Quelantes , Ácido Edético , Escherichia coli/enzimologia , Propriedades de Superfície , Radioisótopos de ÍtrioRESUMO
Short-term external gamma-irradiation (0.1-5 Gy) and long-term (30 days) internal irradiation of rats by everyday intake of 137Cs and 85Sr isotopes into their organisms induced, most probably, irreversible changes in the level of synthetic processes in the rat brain cortex neurons. Synthesis of RNA and proteins decreased significantly after 2h-long irradiation. Irradiation which lasted for 30 days has somewhat shortened the level of reparative DNA synthesis and has almost twice increased RNA synthesis. It was accompanied by changes in the relative activity of RNA-polymerases A, B and C. Doses over 1 Gy have induced an exponential delay of dose-dependent changes in the level of synthetic processes.
Assuntos
Córtex Cerebral/efeitos da radiação , DNA/biossíntese , Proteínas do Tecido Nervoso/biossíntese , RNA/biossíntese , Animais , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , RNA Polimerases Dirigidas por DNA/efeitos da radiação , Relação Dose-Resposta à Radiação , Raios gama , Masculino , Neuroglia/metabolismo , Neuroglia/efeitos da radiação , Neurônios/metabolismo , Neurônios/efeitos da radiação , Ratos , Ratos WistarRESUMO
Stalled elongation complexes of Escherichia coli RNA polymerase were prepared carrying the photo-cross-linkable 8-azido derivative of adenine at the 3'-terminus of the nascent RNA chain. Ultraviolet irradiation of such complexes resulted in the cross-linking of radiolabeled RNA exclusively to the beta' subunit of RNA polymerase. The adduct was mapped between Met932 and Trp1020 in the linear sequence of the beta' polypeptide using specific chemical degradation of the cross-linked species.
Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/enzimologia , RNA Bacteriano/genética , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/metabolismo , Marcadores de Afinidade/metabolismo , Sequência de Aminoácidos , Animais , Azidas/metabolismo , Sítios de Ligação , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/efeitos da radiação , Escherichia coli/genética , Substâncias Macromoleculares , Dados de Sequência Molecular , RNA Bacteriano/metabolismo , Homologia de Sequência do Ácido Nucleico , Transcrição Gênica , Raios UltravioletaAssuntos
RNA Polimerases Dirigidas por DNA/efeitos da radiação , Vírus da Influenza A/efeitos da radiação , Raios Ultravioleta , Vírion/efeitos da radiação , Replicação Viral/efeitos da radiação , Animais , Células Cultivadas/microbiologia , Células Cultivadas/efeitos da radiação , Embrião de Galinha , Relação Dose-Resposta à Radiação , Hemaglutininas Virais/efeitos da radiação , Vírus da Influenza A/enzimologia , Vírus da Influenza A/fisiologia , Neuraminidase/efeitos da radiação , Fatores de Tempo , Vírion/enzimologiaAssuntos
Proteínas de Ligação a DNA/efeitos da radiação , DNA/metabolismo , Lasers , Raios Ultravioleta , Proteínas Virais/efeitos da radiação , Animais , Reagentes de Ligações Cruzadas , DNA/efeitos da radiação , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , RNA Polimerases Dirigidas por DNA/efeitos da radiação , Escherichia coli/metabolismo , Cinética , L-Lactato Desidrogenase/metabolismo , L-Lactato Desidrogenase/efeitos da radiação , Substâncias Macromoleculares , Músculos/enzimologia , Oligodesoxirribonucleotídeos/metabolismo , Coelhos , Relação Estrutura-Atividade , Fagos T/genética , Fagos T/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
Upon irradiation of the RNA polymerase-lacUV5 or deoP1 promoter complex with short wavelength ultraviolet light (lambda less than or equal to 300 nm) the polymerase is covalently crosslinked at an efficiency of greater than 10% to the first transcribed base of the template DNA strand when this is a thymine. The temperature dependence of this RNA polymerase-T+1 photoreaction strongly indicates a relation to the formation of the open complex. It is suggested that open complex formation is preceded or accompanied by a specific contact between the RNA polymerase and the first transcribed base of the DNA template.
Assuntos
RNA Polimerases Dirigidas por DNA/genética , Escherichia coli/genética , Óperon Lac/efeitos da radiação , Regiões Promotoras Genéticas/efeitos da radiação , Timina/efeitos da radiação , Reagentes de Ligações Cruzadas , DNA/efeitos da radiação , RNA Polimerases Dirigidas por DNA/efeitos da radiação , Escherichia coli/enzimologia , Escherichia coli/efeitos da radiação , Fotoquímica , Moldes Genéticos , Transcrição Gênica/efeitos da radiação , Raios UltravioletaRESUMO
Complexes between Bacillus subtilus RNA polymerase and 32P-labeled DNA were irradiated with UV light and digested with nuclease; electrophoresis and autoradiography were used to identify the polymerase subunits cross-linked to DNA. These experiments showed: 1) that cross-linkage of promoter complexes yielded predominantly the beta and sigma subunits; 2) that beta, beta', and sigma were detected in non-promoter complexes; 3) that addition of the delta subunit or high concentrations of NaCl decreased cross-linkage of all subunits, especially the cross-linkage of the sigma subunit in non-promoter complexes and the binding of polymerase at DNA ends; 4) that different patterns of cross-linkage were obtained at 0 degrees C (conditions favoring the formation of closed complexes) and 37 degrees C (conditions favoring the formation of open complexes); and 5) predominantly beta and possibly alpha were cross-linked by irradiation of core-DNA complexes whereas similar experiments with core-delta complexed to DNA showed the efficient cross-linkage of beta' and beta.
Assuntos
Bacillus subtilis/enzimologia , DNA Viral/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Óperon , Raios Ultravioleta , Bacteriófagos/genética , DNA Viral/efeitos da radiação , RNA Polimerases Dirigidas por DNA/efeitos da radiação , Plasmídeos , Cloreto de Sódio/farmacologia , TemperaturaRESUMO
The interaction between the three Drosophila DNA-dependent RNA polymerases (EC 2.7.7.6) and the DNA template or the RNA product was investigated by photochemical cross-linking and binding studies, using RNA polymerase subunits immobilized on nitro-cellulose filters. It can be shown that the two largest subunits are responsible for the binding of the enzymes to both template and newly-synthesized RNA.
Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , DNA/metabolismo , RNA/metabolismo , Animais , DNA/efeitos da radiação , RNA Polimerases Dirigidas por DNA/efeitos da radiação , Drosophila , Fotoquímica , Ligação Proteica/efeitos da radiação , RNA/efeitos da radiaçãoRESUMO
A combined rapid mixing-photocrosslinking technique has been developed to investigate the kinetics of the interaction between Escherichia coli RNA polymerase and T7 DNA. The reactants were rapidly mixed in a modified Durrum stopped-flow apparatus, and the intermediates formed at different stages of the binding process were "frozen" by photocrosslinking with a UV light pulse of 10-mus duration at various times after mixing. The results indicate that the initial binding between RNA polymerase and T7 DNA is a diffusion-controlled reaction. Furthermore, the extents of initial contracts with DNA made with the beta, beta', and sigma subunits of RNA polymerase are roughly proportional to the sizes of these subunits, suggesting that complex formation occurs through random collision between the two reactants. After the initial complex formation, the rate of transfer of polymerase between individual DNA molecules is slow, implying that the polymerase molecules are undergoing predominantly intramolecular transfer during the promoter search. From the kinetic studies of subunit-DNA contacts during RNA polymerase binding to T7 DNA, it can be inferred that the beta, beta', and sigma subunits are directly participating in the promoter search process.
Assuntos
DNA Viral/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/enzimologia , Óperon , Transcrição Gênica , DNA Viral/efeitos da radiação , RNA Polimerases Dirigidas por DNA/efeitos da radiação , Escherichia coli/genética , Cinética , Ligação Proteica , Fagos T/genética , Raios UltravioletaRESUMO
The rapid mixing-photocrosslinking technique, in conjunction with an immunoprecipitation assay developed to measure the change in the distribution of Escherichia coli RNA polymerase molecules bound to T7 DNA, has been applied to investigate the molecular mechanism of promoter search by RNA polymerase. The binding of RNA polymerase to the DNA template can be divided into at least two steps. The initial binding is rapid and occurs at nonspecific sites randomly distributed throughout the DNA molecule. This is followed by a relatively slow promoter search in which RNA polymerase is transferred from nonspecific sites to promoter sites through a series of intramolecular processes. The rate of polymerase loss from a segment of DNA which does not contain promoter sites is a function of the distance from this segment to both the promoter sites and the ends of the DNA molecule. The kinetic data are consistent with a molecular mechanism in which RNA polymerase undergoes a bidirectional linear diffusion along the DNA template to search for the promoter site. This interpretation is supported by the computer simulation which correctly predicts the relative rates of polymerase loss from various DNA segments. The mechanism derived from these studies is in accordance with the notion that the whole DNA molecule serves as an effective sink for trapping and guiding polymerase molecules during promoter search.
Assuntos
DNA Viral/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Óperon , Transcrição Gênica , DNA Viral/efeitos da radiação , RNA Polimerases Dirigidas por DNA/efeitos da radiação , Difusão , Escherichia coli/enzimologia , Cinética , Ligação Proteica , Fagos T/metabolismo , Moldes Genéticos , Raios UltravioletaRESUMO
The first photochemical crosslinking of a protein to a nucleic acid using laser excitation is reported. A single, 120 mJ, 20 ns pulse at 248 nm crosslinks about 10% of bound E. coli RNA polymerase to T7 DNA under the conditions studied. The crosslinking yield depends on mercaptoethanol concentration, and is a linear function of laser intensity. The protein subunits crosslinked to DNA are beta, beta' and sigma.