RESUMEN
Specificity in transcriptional regulation lies in a large part in the specificity of DNA binding by transcription factors. One group of transcription factors which are of great interest for studying transcriptional specificity is the Pax/Homeodomain (Pax/HD) proteins which contain two conserved DNA binding domains, a paired domain (PD) and a Paired-class homeodomain (HD). The Pax/HD proteins can bind to at least three types of specific DNA sequences: the PD binding sites, the dimeric HD binding sites and a composite HD and PD binding site. We propose that Pax/HD proteins regulate different subsets of their target genes through modular binding to one of these three specific sequences. We show that, in a tissue culture system, a member of the Pax/HD family, Paired, is able to activate transcription after binding through either its PD or its HD. The transactivation mediated by one domain does not require DNA binding of the other domain. Furthermore, binding sites specific for the PD of Paired are sufficient to mediate embryonic expression of a reporter gene in a paired-like pattern. The expression of the reporter gene is dependent on wild type paired function and, in a prd mutant background, it can be rescued by an exogenous paired gene encoding a protein whose HD is not able to bind to DNA. Finally, we show that the Paired protein uses differently its C-terminal activation domain when transactivation is mediated through its PD or its HD. These results and recent evidence from other Pax/HD proteins strongly suggest that this class of proteins is able to achieve specific and modular transcriptional regulation through its multiple DNA binding domains.
Asunto(s)
Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica/fisiología , Proteínas de Homeodominio/genética , Factores de Transcripción/genética , Transcripción Genética/fisiología , Animales , Secuencia de Bases , Células Cultivadas , Clonación Molecular , Proteínas de Unión al ADN/biosíntesis , Drosophila , Eliminación de Gen , Genes Reporteros , Proteínas de Homeodominio/biosíntesis , Datos de Secuencia Molecular , Mutagénesis , Factores de Transcripción Paired Box , Factores de Transcripción/biosíntesis , TransgenesRESUMEN
The Drosophila pair-rule gene paired is required for the correct expression of the segment polarity genes wingless, engrailed and gooseberry. It encodes a protein containing three conserved motifs: a homeodomain (HD), a paired domain (PD) and a PRD (His/Pro) repeat. We use a rescue assay in which paired (or a mutated version of paired in which the functions of the conserved motifs have been altered) is expressed under the control of its own promoter, in the absence of endogenous paired, to dissect the Paired protein in vivo. We show that both the HD and the N- terminal subdomain of the PD (PAI domain) are absolutely required within the same molecule for normal paired function. In contrast, the conserved C-terminal subdomain of the PD (RED domain) appears to be dispensable. Furthermore, although a mutation abolishing the ability of the homeodomain to dimerize results in an impaired Paired molecule, this molecule is nonetheless able to mediate a high degree of rescue. Finally, a paired transgene lacking the PRD repeat is functionally impaired, but still able to rescue to viability. We conclude that, while Prd can use its DNA-binding domains combinatorially in order to achieve different DNA-binding specificities, its principal binding mode requires a cooperative interaction between the PAI domain and the homeodomain.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila , Drosophila/embriología , Genes de Insecto , Proteínas de Homeodominio/metabolismo , Animales , Secuencia de Bases , Clonación Molecular , Secuencia Conservada , ADN/metabolismo , Sondas de ADN , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Drosophila/genética , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/química , Proteínas de Homeodominio/genética , Hibridación in Situ , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación Puntual , Reacción en Cadena de la Polimerasa , Eliminación de Secuencia , TransgenesRESUMEN
The TATA box-binding factor TFIID plays a primary role in the process of transcription initiation by RNA polymerase II and its regulation by various gene-specific factors. Here we employ a permuted binding site/gel retardation assay with recombinant yeast and human TFIID to show that this factor induces DNA bending around the TATA element. These results are consistent with the presence of G + C-rich sequence elements flanking the consensus TATA element and led to the recently confirmed suggestion that TFIID interacts with the TATA element via the minor groove. They also raise the possibility that TFIID-induced bending might facilitate promoter interactions of other general factors in the preinitiation complex or interactions between general transcription factors and regulatory factors bound at upstream sites.