GA-binding protein-dependent transcription initiator elements - Effect of helical spacing between polyomavirus enhancer a factor 3(PEA3)/Ets-binding sites on initiator activity

Many eukaryotic RNA polymerase II promoters contain initiator elements which direct accurate transcription in a TATA-independent manner, The PEA3/Ets-binding site (PEA3/EBS) is a common enhancer element in eukaryotic genes and is also found near the transcriptional start sites of many TATA-less promoters. We demonstrate that two PEA3/EBSs driving expression of the luciferase reporter gene, function as a minimal transcriptional initiator element, Maximal levels of transcription was achieved when two PEA3/EBSs, in either orientation, were located on the same face of the DNA helix, and the sites could be separated by up to three helical turns, In vitro transcription start sites directed by PEA3/EBS elements were clustered on either side of the upstream PEA3/EBS and were abolished by immunodepletion of GA-binding protein (GABP) from FM3A cell nuclear extracts. In vivo, co-transfection of GABP alpha and GABP beta expression vectors enhanced reporter gene expression driven from PEA3/EBS initiator elements, Like other initiator elements, the PEA3/EBS elements were activated synergistically by upstream Spl-binding sites. Thus, our results establish GABP as both a transcriptional activator factor and as an initiator factor.