Cooperative binding of GA-binding protein transcription factors to duplicated transcription initiation region repeats of the cytochrome c oxidase subunit IV gene

Abstract

Transcriptional activity of the TATA-less cytochrome c oxidase subunit IV (COXIV) gene promoter depends upon two tandemly repeated sequence elements, each mapping immediately downstream of major loci of transcriptional initiation. In this paper, we demonstrate that binding of the GA-binding protein (GABP) to ets sequence motifs within each repeated unit is required for transcriptional activation of the COXIV promoter. High affinity binding of GABP to the COXIV promoter required both the DNA-binding GABP alpha subunit and the non-DNA-binding GABP beta subunit. Binding of the heteromeric GABP complex to sequences containing two GABP binding sites was shown to have a 10-20-fold greater affinity than to DNA sequences with a single site. GABP binding was necessary for promoter function of a 33-base pair fragment of the COXIV initiation region in transfected 3T3 or COS cells. Binding of GABP to the COXIV initiation region was also required for maximal transcriptional stimulation by an upstream Sp1 binding site. The initiation region was demonstrated to direct accurate transcriptional initiation in vitro, and mutations to the GABP binding sites affected not only transcriptional activity but also initiation site selection. These results indicate that the initiation region repeats of the COXIV promoter may function as GABP-dependent initiator motifs that position mRNA start sites in the absence of a TATA box or other promoter elements.