The chromatin structure specific to the SV40 late transcription elongation complex as well as the occupancy of several sites that bind transcription factors have been examined. These features have been determined by assessing blockage to restriction enzyme digestion. Cleavage specific to the elongation complex has been quantified using ternary complex analysis. This method involves radioactively labeling the complex by in vitro transcription followed by determining the extent of linearization by electrophoresis in an agarose gel. It was found that not only is the origin region devoid of nucleosomes, but there is also no stable factor occupancy at the BglI, SphI, KpnI and MspI restriction enzyme sites within this region. Thus these sites were cleaved to a high degree, meaning that the binding sites for a number of transcription factors, including OBP/TEF-1, TBP, DAP, as well as a proposed positioned nucleosome, are unoccupied in the native viral transcription elongation complex. The absence of these trans-acting factors from their respective binding sites in the elongation complex indicates that they bind only transiently, possibly cycling on and off during the transcription cycle. This finding implies that various forms of transcription complex are assembled and disassembled during transcription and thus supports a 'hit-and-run' model of factor function.
Ammonium Sulfate Cells, Cultured