Structural and Functional Investigation of Promoter Distortion and Opening in the RNA Polymerase II Cleft
by Christian Dienemann
Date of Examination:2018-04-09
Date of issue:2019-02-11
Advisor:Prof. Dr. Patrick Cramer
Referee:Prof. Dr. Patrick Cramer
Referee:Prof. Dr. Ralf Ficner
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Description:Doctoral Thesis
Abstract
English
Transcription of genes is a central process of life that defines how genetic information is used. This determines the metabolic state, morphology and fate of a cell, tissue, organ and the entire organism. In eukaryotes, the most regulated step of transcription is initiation during which RNA polymerase II (Pol II) is specifically recruited to the gene promoter by assembling with transcription factors (TF) –IIA, -IIB, -IID, -IIF, -IIE, -IIH and the co- activator Mediator complex. For initial transcription, the double helix of the promoter DNA of the closed complex (CC) has to be opened in the initially melted region (IMR). The open complex (OC) then provides the single stranded template DNA that can be transcribed. Promoter opening was shown to require the DNA translocase activity of TFIIH; however, spontaneous DNA melting could also be observed. The molecular pathway of TFIIH dependent and spontaneous promoter melting is still unknown due to a lack of high resolution structural studies targeting intermediates of the CC-to-OC transition. Here I report the structure of a new transcription initiation intermediate just before promoter DNA opening at ~5 Å resolution. This structure reveals closed distorted promoter DNA in the Pol II active center cleft. The distortion is induced by the closed Pol II clamp domain and stabilized by structural elements of TFIIF and TFIIE. The distorted DNA shows a helical axis offset and underwinding of the closed promoter DNA that weaken the DNA and prime it for melting. I further present in vivo and in vitro data showing that the DNA duplex stability of the IMR determines whether DNA spontaneously melts after distortion or whether it remains closed and requires TFIIH activity to be melted. By systematic comparison of Pol I and Pol III promoters with the Pol II system, I find that promoters of the other nuclear RNA polymerases also contain a weak DNA duplex in their IMR. This explains why they do not need a TFIIH homologue or ATP-hydrolysis to open promoter DNA. This suggests a unified mechanism of promoter DNA melting that involves DNA distortion by clamp closure and subsequent melting of the IMR. Taken together, these results greatly improve our understanding of the molecular mechanism of promoter DNA melting by nuclear RNA polymerases and for the first time provide a general mechanism for promoter opening.
Keywords: transcription; RNA polymerase II; TFIIH; Ssl2; XPB; promoter opening; DNA distortion