User:Anthony Salvagno/Notebook/Research/2010/07/29/Synergistic action of RNA Polymerases in overcoming nucleosomal barriers

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by Jing Jin, et al (an MD Wang paper)

I'm going to try and get through 2-3 Wang papers today. Probably just two. Hopefully not just one. Yesterday I only summarized one paper but I read another to find out that even though OT was in the title there was no mention of the tweezers in the paper. Strange. Anyways, on with it.

Summary and Comments

They first identified the unzipping signature of RNAP. First they created an unzipping construct with a promoter and stalled the polymerase 20b upstream via UTP depletion. Next they tried the same technique on nucleosomal DNA. The DNA was setup similarly to the previous step only they added a 601 nucleosome positioning element (NPE) which I suppose is a sequence of DNA that binds to a nucleosome. They unzipped the RNAP and the nucleosome to get the unzipping sig. of both. They then did some experiments where they allowed the RNAP to continue elongation, unzipping at different points along the way.

They restarted elongation without undergoing reinitiation. They did this by introducing another DNA strand with a promoter on it. I don't understand how this works at all. They proved it works by showing that after ~30 min without competitor DNA more and more RNAPs would show up at their +20nt stall site, while with the competitor there were none. I understand the data, but I don't understand the mechanism as to why that works. Here is my best guess: the presence of the competitor drives new polymerases to that promoter for initiation allowing the stalled polymerases on the experimental strand to proceed uninhibited.

Then they unzipped but had a hard time distinguishing the RNAP from the nucleosome so they added heparin to release the nucleosome so they could observe the RNAP's progression through the nucleosome (at specific time points). They claim that the heparin doesn't do anything to the RNAP but their supplementary data shows (to me) that it increases the force required to unbind the polymerase.