FATE OF LINEAR AND SUPERCOILED MULTINUCLEOSOMIC TEMPLATES DURING TRANSCRIPTION

Electron microscopy was used to monitor the fate of reconstituted nucl eosome cores during in vitro transcription of long linear and supercoi led multinucleosomic templates by the prokaryotic T7 RNA polymerase an d the eukaryotic RNA polymerase II. Transcription by T7 RNA polymerase disrupted the nucleosomal configuration in the transcribed region, wh ile nucleosomes were preserved upstream of the transcription initiatio n site and in front of the polymerase. Nucleosome disruption was indep endent of the topology of the template, linear or supercoiled, and of the presence or absence of nucleosome positioning sequences in the tra nscribed region, In contrast, the nucleosomal configuration was preser ved during transcription from the vitellogenin B1 promoter with RNA po lymerase II in a rat liver total nuclear extract, However, the persist ence of nucleosomes on the template was not RNA polymerase II-specific , but was dependent on another activity present in the nuclear extract , This was demonstrated by addition of the extract to the T7 RNA polym erase transcription reaction, which resulted in retention of the nucle osomal configuration, This nuclear activity, also found in HeLa cell n uclei, is heat sensitive and could not be substituted by nucleoplasmin , chromatin assembly factor (CAF-I) or a combination thereof, Altogeth er, these results identify a novel nuclear activity, called herein tra nscription-dependent chromatin stabilizing activity I or TCSA-I, which may be involved in a nucleosome transfer mechanism during transcripti on.