Question

You are studying the function of phosphorylation of TFIIH with extracts from yeast. You can add these extracts to dsDNA and have transcription occur in vitro (if you also add rNTPs). Your research team has created an inhibitor that specifically prevents TFIIH from phosphorylating the CTD of RNA pol II. You decide to measure the size of the transcription bubble with and without the inhibitor. Develop or find an assay that would measure the size of the bubble and describe it, with controls. How big would the bubble be with and without the inhibitor?

Answer 1

Taken from J. Mol. Biol. (2001) 308, 465-475

Pyrrolo-dC is a cytidine analog that binds to guanine and has a fluorescence emission maxima at 460 nm. What makes it useful in this context is that it has reduced fluorescence in duplex DNA than in ssDNA.

We first prepare an assay of DNA strands with increasing number of pyrrolo-dC-guanine pairs sandwiched between A-T pairs.

Lowercase 'c' stands for pyrrolo-dC

AAAAAAAcAAAAAAA

AAAAAAAccAAAAAA  

AAAAAAcccAAAAAA

*

**

***

AAccccccccccccccAA

Adding TFIIH+RNA pol II+DNA in an in-vitro setup and measuring the fluorescence of the DNA strand will give a plateau-shaped curve of fluorescence intensity versus time. This happens because initially we have pyrrolo-dC in form of c-G pairs when intensity is low; intensity abruptly increases as RNA pol II unwinds this part of the DNA and the c-G pairs break, and intensity value finally falls back again as transcription bubble passes over and duplex DNA reforms.

Transcription bubble time = 0 Low fluorescence RNA pol I time = 1 High fluorescence time = 2 Low fluorescence (again)

The intensity profile for a DNA strand having two c-G pairs sandwiched between A-T s would be approximately double of the intensity profile for DNA strand having only one c-G pair, for all time instants. Similar is the comparison between DNA strands containing 20 and 21 c-G pairs, respectively. But if the size of transcription bubble is, let's say, 10 base pairs, then the 'width' of the plateau in the intensity vs time curve would be zero (like a triangular hill, not a plateau). This would happen because the instant of maximum intensity (when the entire transcription bubble is composed of c-G pairs) occurs only once. Contrast with the scenario where number of c-G pairs is greater than the transcription bubble, when we get maximum intensity for all the time in which RNA pol II is unwinding the c-G region.

Zero c-G pairs inside bubble -Zero C-G pairs inside bubbles -Zero c-G pairs inside bubbles Less than 10 pairs inside bubble Less than 5 pairs inside bubble Less than 20pairs inside bubble 10 C-G pairs inside bubble All c-G pairs inside bubble | 10 c-G pairs inside bubble Intensity 二区 Intensity Intensity Time Time Time Intensity profile for DNA containing 5c-G pairs Intensity profile for DNA containing20 c-G pairs Intensity profile for DNA containing10 c-G pairs

As for the last question, I myself am not sure. But a sentence taken from the abstract of Mol Cell. 2014 May 22;54(4):601-12 states that inhibiting kinase ability of TFIIH prevents promoter escape, and a sentence taken from the abstract of Biochem J. 2014 Oct 1;463(1):135-44 states that promoter escape causes collapsing of the transcription bubble. Putting two and two together, we see that the transcription bubble would be larger in presence of the kinase domain inhibitor and smaller when TFIIH can phosphorylate RNA pol II CTD.