MINOR-GROOVE CONTACTS ARE ESSENTIAL FOR AN INTERACTION OF THE HUMAN CYTOMEGALOVIRUS IE2 PROTEIN WITH ITS DNA TARGET

The 86 kDa immediate early-2 protein (IE2, IE86) of human cytomegalovi rus (HCMV) is a multifunctional polypeptide that can regulate gene exp ression both positively and negatively. In particular, it represses it s own mRNA synthesis by binding directly to a sequence element, termed cis repression signal (CRS), that is located between the TATA box and the transcriptional start site of the major IE enhancer/promoter of H CMV. Here, we provide evidence that IE86, unlike most sequence-specifi c DNA-binding proteins, interacts primarily within the minor groove of the DNA helix, This was shown by hydroxyl radical and methylation int erference assays. In addition, binding studies with inosine-substitute d oligonucleotides which have an altered major groove morphology witho ut changing the surface of the minor groove, confirmed the results obt ained in interference analyses. This establishes IE86 as a member of a small group of DNA binding proteins that interact with A - T rich seq uences within the minor groove and which also includes the TATA-box bi nding protein TBP. Remarkably, IE86 and TBP are able to bind simultane ously in an immediate vicinity at the major IE enhancer/promoter of HC MV. As minor groove binding proteins are known to bend DNA heavily thi s could contribute to the observed negative regulation of transcriptio n by IE86.