DIRECT INTERACTION OF THE HUMAN CYTOMEGALOVIRUS IE86 PROTEIN WITH THECIS REPRESSION SIGNAL DOES NOT PRECLUDE TBP FROM BINDING TO THE TATA BOX

The human cytomegalovirus major immediate-early gene encodes several p rotein isoforms which autoregulate the major immediate-early promoter (MIEP). One of these isoforms, the IE86 protein, represses the MIEP th rough a DNA sequence located between the TATA box and the transcriptio n initiation site, designated the cis repression signal (crs). Through mutational analysis, amino acid domains within IE86 responsible for b inding the crs element were located at the C terminus. Mutation of the putative zinc finger domain, which precluded IE86 from binding DNA, c onverted the protein from a repressor of MIEP transcription into an ac tivator. DNase I protection analysis demonstrated that the IE86 footpr int overlapped the sequence protected by the TATA-binding protein (TBP ). Investigation of whether IE86 was able to displace TBP from DNA rev ealed that both proteins could bind DNA simultaneously. However, highe r concentrations of IE86 were required to obtain protection of the crs element in the presence of prebound TBP. Similarly, higher concentrat ions of TBP were required to obtain protection in the presence of preb ound IE86. These observations indicate that steric hinderance impairs but does not prevent both proteins from binding DNA synchronously.