Asunto(s)
ADN/química , Dietil Pirocarbonato , Conformación de Ácido Nucleico , Permanganato de Potasio , Acanthamoeba/enzimología , Acanthamoeba/genética , Animales , Secuencia de Bases , ADN/aislamiento & purificación , Electroforesis en Gel de Poliacrilamida/métodos , Indicadores y Reactivos , Reacción en Cadena de la Polimerasa/métodos , Regiones Promotoras Genéticas , ARN Polimerasa I/genética , Transcripción GenéticaRESUMEN
Ribosomal RNA transcription initiation requires the melting of DNA to form an open complex, formation of the first few phosphodiester bonds, commencement of RNA polymerase I movement along the DNA, clearance of the promoter, and the formation of a steady-state ternary elongation complex. We examined DNA melting and promoter clearance by using potassium permanganate, diethylpyrocarbonate and methidiumpropylEDTA.Fe(II) footprinting. In combination, these methods demonstrated: (1) TIF-IB and RNA polymerase I are the only proteins required for formation of an initial approximately 9 base-pair open promoter region. This finding contradicts earlier results using diethylpyrocarbonate alone, which suggested an RNA synthesis requirement for stable melting. (2) DNA melting is temperature-dependent, with a tm between 15 and 20 degrees C. (3) Temperature-dependency of melting, as well as stalling the polymerase at sites close to the transcription start site revealed that the melted DNA region initially opens upstream of the transcription initiation site, and enlarges in a downstream direction coordinate with initiation, eventually attaining a steady-state transcription bubble of approximately 19 base-pairs. (4) The RNA-DNA hybrid protects the template DNA from single-strand footprinting reagents. The hybrid is 9 bp in length, consistent with the longer hybrid estimated by some for the Escherichia coli polymerase and with the hybrids estimated for eukaryotic polymerases II and III.
Asunto(s)
ADN/química , ADN/metabolismo , Desnaturalización de Ácido Nucleico/genética , Proteínas del Complejo de Iniciación de Transcripción Pol1 , Regiones Promotoras Genéticas/genética , ARN Polimerasa I/metabolismo , Transcripción Genética/genética , Acanthamoeba/enzimología , Acanthamoeba/genética , Animales , Emparejamiento Base/genética , Secuencia de Bases , Sitios de Unión , ADN/genética , Huella de ADN , Proteínas de Unión al ADN/metabolismo , Dietil Pirocarbonato/metabolismo , Ácido Edético/análogos & derivados , Ácido Edético/metabolismo , Genes de ARNr/genética , Datos de Secuencia Molecular , Mutación/genética , Ácidos Nucleicos Heterodúplex/genética , Permanganato de Potasio/metabolismo , Unión Proteica , ARN Ribosómico/biosíntesis , ARN Ribosómico/química , ARN Ribosómico/genética , ARN Ribosómico/metabolismo , Sarcosina/análogos & derivados , Sarcosina/farmacología , Temperatura , Moldes Genéticos , Factores de Transcripción/metabolismo , Transcripción Genética/efectos de los fármacosRESUMEN
The effects of substituting (2S,3S)-beta-methylphenylalanine (S-beta MeF) or (2S,3R)-beta-methylphenylalanine (R-beta MeF) for the Phe7 and/or Phe8 residues of the tachykinin substance P (SP, RPKPQQFFGLM-NH2) upon the ability of SP to stimulate contraction of the rabbit iris smooth muscle were investigated. The eight beta MeF-containing SP analogs (four monosubstituted analogs, four disubstituted analogs) 1-8 were synthesized and found to be agonsts of SP in the smooth muscle contraction assay, having EC50 values ranging from 0.15 to 10.0 nM. Three analogs are significantly more active than SP [8R-(beta MeF)SP (4), 7S,8S-(beta MeF)2SP (5), and 7R,8S-(beta MeF)2SP (6)], three analogs are approximately equipotent with SP [7S-(beta MeF)SP (1), 7R-(beta MeF)SP (2), and 7S,8R-(beta MeF)2SP (8)], and two analogs are significantly less active than SP [8S-(beta MeF)SP (3) and 7R,8R-(beta MeF)2SP (7)]. The effects of the beta MeF substitutions upon the activity of SP are not additive and cannot be explained using simple conformational models which focus only on the side chain conformations of the beta MeF residues. It is postulated that the beta MeF residues induce minor distortions in the peptide backbone with resultant consequences upon peptide-receptor binding which are not dictated soley by the side chain conformations. This idea is consistent with 1H-NMR data for the monosubstituted analogs 1-4, which imply that the beta MeF substitutions cause slight distortions in the peptide backbone and that the beta MeF side chains are assuming trans or gauche(-) conformations.