TFIIB is only ∼9 Å away from the 5'-end of a trimeric RNA primer in a functional RNA polymerase II preinitiation complex

• Published in: PloS one Vol. 10; no. 3; p. e0119007
• Main Authors: Bick, Matthew J, Malik, Sohail, Mustaev, Arkady, Darst, Seth A
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 16-03-2015; Public Library of Science (PLoS)
• Subjects: Bacteria - metabolism; Biophysics; Catalytic Domain; Crosslinking; Crystallography; Deoxyribonucleic acid; DNA; DNA-directed RNA polymerase; E coli; Eukaryotic Cells - metabolism; Laboratories; Models, Molecular; Mutation; Promoter Regions, Genetic; Ribonucleic acid; RNA; RNA - chemistry; RNA - metabolism; RNA polymerase; RNA polymerase II; RNA Polymerase II - chemistry; RNA Polymerase II - metabolism; Sigma Factor - chemistry; Sigma Factor - metabolism; Site selection; Structural analysis; Transcription Factor TFIIB - chemistry; Transcription Factor TFIIB - metabolism; Transcription factors; Transcription initiation factor TFIIB; Transcription, Genetic; New York; United States > US
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0119007

Recent X-ray crystallographic studies of Pol II in complex with the general transcription factor (GTF) IIB have begun to provide insights into the mechanism of transcription initiation. These structures have also shed light on the architecture of the transcription preinitiation complex (PIC). However, structural characterization of a functional PIC is still lacking, and even the topological arrangement of the GTFs in the Pol II complex is a matter of contention. We have extended our activity-based affinity crosslinking studies, initially developed to investigate the interaction of bacterial RNA polymerase with σ, to the eukaryotic transcription machinery. Towards that end, we sought to identify GTFs that are within the Pol II active site in a functioning PIC. We provide biochemical evidence that TFIIB is located within ∼9 Å of the -2 site of promoter DNA, where it is positioned to play a role in de novo transcription initiation.