SPECIFIC BINDING OF RNA-POLYMERASE-II TO THE HUMAN-IMMUNODEFICIENCY-VIRUS TRANSACTIVATING REGION RNA IS REGULATED BY CELLULAR COFACTORS ANDTAT

The regulation of human immunodeficiency virus type 1 (HIV-1) gene exp ression in response to Tat is dependent on an element downstream of th e HIV-1 transcriptional initiation site designated the trans-activatin g region (TAR), TAR forms a stable stem-loop RNA structure in which a 3-nt bulge structure and a 6-nt loop structure are important for Tat a ctivation. In the absence of Tat, the HIV-1 promoter generates so-call ed short or nonprocessive transcripts terminating at +60, while in the presence of Tat the synthesis of these short transcripts is markedly decreased and transcripts that extend through the 9.0-kb HIV-1 genome are synthesized, Tat effects on transcriptional elongation are likely due tb alterations in the elongation properties of RNA polymerase II, In this study we demonstrated that a set of cellular cofactors that mo dulate the binding of the cellular protein TRP-185 to the TAR RNA loop sequences also functioned to markedly stimulate the specific binding of hypophosphorylated (IIa) and hyperphosphorylated (IIo) RNA polymera se II to TAR RNA. The concentrations of RNA polymerase II required for this interaction with TAR RNA were similar to those required to initi ate in vitro transcription from the HIV-1 long terminal repeat, RNA ge l retardation analysis with wild-type and mutant TAR RNAs indicated th at the TAR RNA loop and bulge sequences were critical for the binding of RNA polymerase II, The addition of wild-type but not mutant Tar pro tein to gel retardation analysis with TAR RNA and RNA polymerase II re sulted in the loss of binding of RNA polymerase II binding to TAR RNA, These results suggest that Tat may function to alter RNA polymerase I I, which is paused due to its binding to HIV-1 TAR RNA with resultant stimulation of its transcriptional elongation properties.