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.