RESUMEN
p270 is an integral member of human SWI-SNF complexes, first identified through its shared antigenic specificity with p300 and CREB binding protein. The deduced amino acid sequence of p270 reported here indicates that it is a member of an evolutionarily conserved family of proteins distinguished by the presence of a DNA binding motif termed ARID (AT-rich interactive domain). The ARID consensus and other structural features are common to both p270 and yeast SWI1, suggesting that p270 is a human counterpart of SWI1. The approximately 100-residue ARID sequence is present in a series of proteins strongly implicated in the regulation of cell growth, development, and tissue-specific gene expression. Although about a dozen ARID proteins can be identified from database searches, to date, only Bright (a regulator of B-cell-specific gene expression), dead ringer (a Drosophila melanogaster gene product required for normal development), and MRF-2 (which represses expression from the cytomegalovirus enhancer) have been analyzed directly in regard to their DNA binding properties. Each binds preferentially to AT-rich sites. In contrast, p270 shows no sequence preference in its DNA binding activity, thereby demonstrating that AT-rich binding is not an intrinsic property of ARID domains and that ARID family proteins may be involved in a wider range of DNA interactions.
Asunto(s)
Proteínas de Unión al ADN/química , Proteínas Nucleares , Factores de Transcripción/química , Factores de Transcripción/fisiología , Secuencia de Aminoácidos , Secuencia de Bases , Northern Blotting , ADN Complementario/metabolismo , Humanos , Modelos Genéticos , Datos de Secuencia Molecular , Familia de Multigenes , Mutación , Biosíntesis de Proteínas , Proteínas Recombinantes de Fusión/metabolismo , Análisis de Secuencia de Proteína , Homología de Secuencia de Aminoácido , Distribución Tisular , Factores de Transcripción/genéticaRESUMEN
p300 and the closely related CREB binding protein (CBP) are transcriptional adaptors that are present in intracellular complexes with TATA binding protein (TBP) and bind to upstream activators including p53 and nuclear hormone receptors. They have intrinsic and associated histone acetyltransferase activity, suggesting that chromatin modification is an essential part of their role in regulating transcription. Detailed characterization of a panel of antibodies raised against p300/CBP has revealed the existence of a 270-kDa cellular protein, p270, distinct from p300 and CBP but sharing at least two independent epitopes with p300. The subset of p300/CBP-derived antibodies that cross-reacts with p270 consistently coprecipitates a series a cellular proteins with relative molecular masses ranging from 44 to 190 kDa. Purification and analysis of various proteins in this group reveals that they are components of the human SWI/SNF complex and that p270 is an integral member of this complex.
Asunto(s)
Proteínas Nucleares/metabolismo , Transactivadores , Factores de Transcripción/análisis , Factores de Transcripción/metabolismo , Adenosina Trifosfatasas/metabolismo , Secuencia de Aminoácidos , Complejo Antígeno-Anticuerpo/metabolismo , Proteína de Unión a CREB , ADN Helicasas , Proteínas de Unión al ADN/metabolismo , Mapeo Epitopo , Células HeLa , Humanos , Sustancias Macromoleculares , Datos de Secuencia Molecular , Peso Molecular , Proteína de Unión a TATA-Box , Factores de Transcripción/químicaRESUMEN
Overproduction of the alpha subunit of RNA polymerase in Escherichia coli resulted in inhibition of transcription of two osmoregulated porin genes, ompF and ompC, but not of constitutively expressed housekeeping genes. Overproduction of the sigma subunit did not have any inhibitory effects. The specific inhibitory effect of the alpha subunit was also found to depend upon the OmpR protein, the transcriptional activator for ompF and ompC. These results are in general agreement with other biochemical and genetic evidence suggesting that the alpha subunit is the subunit of RNA polymerase that directly interacts with certain transcriptional activators to initiate transcription.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/genética , ARN Polimerasas Dirigidas por ADN/química , Escherichia coli/enzimología , Regulación Bacteriana de la Expresión Génica/genética , Proteínas de la Membrana Bacteriana Externa/biosíntesis , ARN Polimerasas Dirigidas por ADN/metabolismo , Electroforesis en Gel de Poliacrilamida , Escherichia coli/genética , Genes Bacterianos/genética , ARN Bacteriano/análisis , Transcripción GenéticaRESUMEN
D55Q-T83A and D55Q-G94S, two pseudorevertants of the D55Q mutant OmpR, an Escherichia coli transcriptional activator, were isolated previously by R. Brissette, K. Tsung, and M. Inouye (J. Bacteriol. 173:3749-3755, 1991). These pseudorevertant OmpR proteins were purified and examined for their function as transcriptional activators in a cell-free system with an ompF DNA fragment. These proteins were transcriptionally active, even after acid treatment, whereas the wild-type OmpR was completely inactive after the same treatment. Phosphorylation of acid-treated wild-type OmpR with an EnvZ11 membrane fraction and ATP restored transcriptional activity, whereas the activities of the mutant OmpR proteins did not change after phosphorylation.