RESUMEN
A negative regulatory element (NRE) is located immediately upstream of the upstream regulatory sequence of core promoter and second enhancer of human hepatitis B virus (HBV). NRE represses the transcription activation function of the upstream regulatory sequence of core promoter and the second enhancer. In this study, we described the cloning and characterization of an NRE-binding protein (NREBP) through expression cloning. NREBP cDNA is 8266 nucleotides in size and encodes a protein of 2386 amino acids with a predicted molecular mass of 262 kDa. Three previously described cDNAs, DBP-5, SONB, and SONA, are partial sequence and/or alternatively spliced forms of NREBP. The genomic locus of the NREBP/SON gene is composed of 13 exons and 12 introns. The endogenous NREBP protein is localized in the nucleus of human hepatoma HuH-7 cells. Antibody against NREBP protein can specifically block the NRE binding activity present in fractionated nuclear extracts in gel shifting assays, indicating that NREBP is the endogenous nuclear protein that binds to NRE sequence. By polymerase chain reaction-assisted binding site selection assay, we determined that the consensus sequence for NREBP binding is GA(G/T)AN(C/G)(A/G)CC. Overexpression of NREBP enhances the repression of the HBV core promoter activity via NRE. Overexpression of NREBP can also repress the transcription of HBV genes and the production of HBV virions in a transient transfection system that mimics the viral infection in vivo.
Asunto(s)
Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/fisiología , Regulación Viral de la Expresión Génica , Virus de la Hepatitis B/genética , Secuencia de Bases , Sitios de Unión , Clonación Molecular , Secuencia de Consenso , Humanos , Antígenos de Histocompatibilidad Menor , Datos de Secuencia Molecular , ARN Mensajero/biosíntesis , Proteínas Represoras/genética , Proteínas Represoras/fisiología , Elementos de Respuesta , Homología de Secuencia de Aminoácido , Distribución Tisular , Células Tumorales Cultivadas , Replicación ViralRESUMEN
The Klebsiella pneumoniae mdcR gene, which encodes a LysR-type regulator, was overexpressed in Escherichia coli. Purified MdcR was found to bind specifically to the control region of either the malonate decarboxylase (mdc) genes or mdcR. We have also demonstrated that MdcR is an activator of the expression of the mdc genes, whereas it represses the transcription of the putative control region of mdcR, PmdcR, indicating a negative autoregulatory control.
