A cis-acting element in Rous sarcoma virus long terminal repeat required for promoter repression by HeLa nuclear...

HeLa cell basic nuclear protein (p21), which represses Rous sarcoma virus long terminal repeat (RSV LTR) promoter activity, diminished v-src expression and the appearance at permissive temperature of the transformed phenotype in tsRSVLA23 Rat-1, a cell line transformed with a temperature-sensitive mutant of RSV. Nuclear run-on analyses using COS-1 cells cotransfected with p21 cDNA and chloramphenicol acetyltransferase reporter indicated that p21 inhibits transcription initiation by targeting a region in the RSV LTR promoter between positions -108 and -85 upstream of the cap site. Insertion of this 24-base pair sequence in place of one of the 72-base pair enhancers in the SV40 early promoter rendered it sensitive to p21 repression. Electrophoretic mobility shift assays using a synthetic oligomer corresponding to the 24-base pair LTR promoter element revealed that p21 altered the pattern of protein.DNA complex formation apparently without binding DNA directly. Complex formation assayed by UV cross-linking and DNA affinity chromatography indicated further that a cellular factor which can interact with this element was decreased in cells transfected with p21 expression plasmid. The results indicate that p21 repression of RSV LTR is mediated by a cis-acting element and may occur by alteration of protein complexes formed on this promoter element.