SITE-SPECIFIC INHIBITION OF RNA POLYMERASE-II PREINITIATION COMPLEX ASSEMBLY BY HUMAN CYTOMEGALOVIRUS IE86 PROTEIN

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 (UL122, IE2), is a DNA -binding protein that represses the MIEP through its cognate recogniti on sequence (designated the cis repression signal [crs]) located betwe en the TATA box and the initiation site of transcription. Purified rec ombinant IE86 protein was shown to repress MIEP transcription in vitro , in a cis-acting mediated pathway, with nuclear extracts from HeLa S3 , U373-MG, and primary human foreskin fibroblast cells. Repression of the MIEP by IE86 was shown by two criteria to be dependent on the dire ct interaction of IE86 with the crs element. Core promoter constructs containing essentially the MIEP TATA box and crs element were also spe cifically repressed by IE86 but not by a mutant IE86 protein, indicati ng the general transcription machinery as the target for IE86 repressi on. Kinetic and template commitment experiments demonstrated that IE86 affects preinitiation complex formation but not the rate of reinitiat ion. Sarkosyl inhibition experiments further revealed that IE86 was un able to effect repression by either disassembling or preventing the el ongation of a preexisting transcription complex. Further, the ability of IE86 to interact with the DNA-binding subunit of TFIID was shown no t to be required for repression. These functional protein-DNA and prot ein-protein interaction experiments demonstrate that IE86 specifically interferes with the assembly of RNA polymerase II preinitiation compl exes. The biological significance of these results and the precise mec hanism by which IE86 represses transcription are discussed.