Mechanism of enhancer action over a large distance and transcription through the nucleosome by RNA polymerase II.

摘要

Enhancer, a short regulatory DNA sequence composed of several binding sites for activator proteins, can activate transcription from its target promoter in a distance and orientation independent manner. The mechanism of enhancer action over a large distance is investigated in this work. A single nucleosome consists a strong barrier for RNA polymerase II (pol II) transcription in vitro. Role of CTD phosphorylation in transcription through nucleosome is analysis with end-inititated pol II.; Activation of the glnAp2 promoter by the NtrC-dependent σ 54-dependent enhancer in E. coli was analyzed using a purified system supporting multiple-round transcription in vitro . Our results suggest that the enhancer-promoter interaction and the initiation complex must be formed de novo during every round of transcription. No protein remained bound to the promoter after RNA polymerase escaped into elongation. The rate of initiation during the first and subsequent rounds of transcription were very similar, suggesting that there was no functional “memory” facilitating multiple rounds of transcription. Our data also suggest that DNA supercoiling is an essential requirement for enhancer action over a large distance (2500 bp) but not over a short distance (110 bp). DNA supercoiling facilitates functional enhancer-promoter communication over a large distance, probably by bringing the enhancer and promoter into close proximity.; End-initiated transcription of a 256 bp template containing a single uniquely positioned nucleosome by yeast and calf thymus pol II was analyzed in vitro. The nucleosome-specific pausing pattern is similar to the pattern observed by yeast RNA polymerase III, but clearly different with the pattern from promoter-initiated and assembled pol II. The rates of transcription through the nucleosome by phosphorylated (IIO) and non-phosphorylated (IIA) forms of calf thymus pol II are very similar. This suggests that CTD phosphorylation is not sufficient to facilitate transcription through the nucleosome by end-initiated pol II.