The human cytomegalovirus major immediate-early gene encodes several protein 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 recognition sequence (designated the cis repression signal [crs]) located between the TATA box and the initiation site of transcription. Purified recombinant 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 direct interaction of IE86 with the crs element. Core promoter constructs containing essentially the MIEP TATA box and crs element were also specifically repressed by IE86 but not by a mutant IE86 protein, indicating the general transcription machinery as the target for IE86 repression. Kinetic and template commitment experiments demonstrated that IE86 affects preinitiation complex formation but not the rate of reinitiation. Sarkosyl inhibition experiments further revealed that IE86 was unable to effect repression by either disassembling or preventing the elongation of a preexisting transcription complex. Further, the ability of IE86 to interact with the DNA-binding subunit of TFIID was shown not to be required for repression. These functional protein-DNA and protein-protein interaction experiments demonstrate that IE86 specifically interferes with the assembly of RNA polymerase II preinitiation complexes. The biological significance of these results and the precise mechanism by which IE86 represses transcription are discussed.
ASJC Scopus subject areas
- Insect Science