THE CELLULAR FACTOR TRP-185 REGULATES RNA-POLYMERASE-II BINDING TO HIV-1 TAR RNA

Activation of HIV-1 gene expression by the transactivator Tat is depen dent on an RNA regulatory element located downstream of the transcript ion initiation site known as TAR. To characterize cellular factors tha t bind to TAR RNA and are involved in the regulation of HIV-1 transcri ption, HeLa nuclear extract was fractionated and RNA gel-retardation a nalysis was performed. This analysis indicated that only two cellular factors, RNA polymerase II and the previously characterized TAR RNA lo op binding protein TRP-185, were capable of binding specifically to TA R RNA. To elucidate the function of TRP-185, it was purified from HeLa nuclear extract, amino acid microsequence analysis was performed and a cDNA encoding TRP-185 was isolated, TRP-185 is a novel protein of 16 21 amino acids which contains a leucine zipper and potentially a novel RNA binding moth. In gel-retardation assays, the binding of both reco mbinant TRP-185 and RNA polymerase II was dependent on the presence of an additional group of proteins designated cellular cofactors. Both t he TAR RNA loop and bulge sequences were critical for RNA polymerase L I binding, while TRP-185 binding was dependent only on TAR RNA loop se quences. Since binding of TRP-185 and RNA polymerase II to TAR RNA was found to be mutually exclusive, our results suggest that TRP-185 may function either alone or in conjunction with Tat to disengage RNA poly merase II which is stalled upon binding to nascently synthesized TAR R NA during transcriptional elongation.