title: Architecture of the RNA polymerase II-TFIIF complex revealed by cross-linking and mass spectrometry.
abstract: Higher-order multi-protein complexes such as RNA polymerase II (Pol II) complexes with transcription initiation factors are often not amenable to X-ray structure determination. Here, we show that protein cross-linking coupled to mass spectrometry (MS) has now sufficiently advanced as a tool to extend the Pol II structure to a 15-subunit, 670 kDa complex of Pol II with the initiation factor TFIIF at peptide resolution. The N-terminal regions of TFIIF subunits Tfg1 and Tfg2 form a dimerization domain that binds the Pol II lobe on the Rpb2 side of the active centre cleft near downstream DNA. The C-terminal winged helix (WH) domains of Tfg1 and Tfg2 are mobile, but the Tfg2 WH domain can reside at the Pol II protrusion near the predicted path of upstream DNA in the initiation complex. The linkers between the dimerization domain and the WH domains in Tfg1 and Tfg2 are located to the jaws and protrusion, respectively. The results suggest how TFIIF suppresses non-specific DNA binding and how it helps to recruit promoter DNA and to set the transcription start site. This work establishes cross-linking/MS as an integrated structure analysis tool for large multi-protein complexes.
authors.affiliation: Wellcome Trust Centre for Cell Biology, Institute of Cell Biology, The University of Edinburgh, Edinburgh, UK.
authors.forename: Zhuo Angel
authors.initials: ZA
authors.lastname: Chen
authors.forename: Anass
authors.initials: A
authors.lastname: Jawhari
authors.forename: Lutz
authors.initials: L
authors.lastname: Fischer
authors.forename: Claudia
authors.initials: C
authors.lastname: Buchen
authors.forename: Salman
authors.initials: S
authors.lastname: Tahir
authors.forename: Tomislav
authors.initials: T
authors.lastname: Kamenski
authors.forename: Morten
authors.initials: M
authors.lastname: Rasmussen
authors.forename: Laurent
authors.initials: L
authors.lastname: Lariviere
authors.forename: Jimi-Carlo
authors.initials: JC
authors.lastname: Bukowski-Wills
authors.forename: Michael
authors.initials: M
authors.lastname: Nilges
authors.forename: Patrick
authors.initials: P
authors.lastname: Cramer
authors.forename: Juri
authors.initials: J
authors.lastname: Rappsilber
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D003432:Cross-Linking Reagents; D004271:DNA, Fungal / Q000235:genetics; D006801:Humans; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D046912:Multiprotein Complexes; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D021122:Protein Subunits; D012319:RNA Polymerase II / Q000737:chemistry* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics; D040681:Structural Homology, Protein; D019473:Transcription Factors, TFII / Q000737:chemistry* / Q000235:genetics
chemical_list: D003432:Cross-Linking Reagents; D004271:DNA, Fungal; D046912:Multiprotein Complexes; D021122:Protein Subunits; D029701:Saccharomyces cerevisiae Proteins; D019473:Transcription Factors, TFII; D012319:RNA Polymerase II; C056604:transcription factor TFIIF