title: Crosslink Mapping at Amino Acid-Base Resolution Reveals the Path of Scrunched DNA in Initial Transcribing Complexes.
abstract: RNA polymerase binds tightly to DNA to recognize promoters with high specificity but then releases these contacts during the initial stage of transcription. We report a site-specific crosslinking approach to map the DNA path in bacterial transcription intermediates at amino acid and nucleotide resolution. After validating the approach by showing that the DNA path in open complexes (RPO) is the same as in high-resolution X-ray structures, we define the path following substrate addition in "scrunched" complexes (RPITC). The DNA bulges that form within the transcription bubble in RPITC are positioned differently on the two strands. Our data suggest that the non-template strand bulge is extruded into solvent in complexes containing a 5-mer RNA, whereas the template strand bulge remains within the template strand tunnel, exerting stress on interactions between the β flap, β' clamp, and σ3.2. We propose that this stress contributes to σ3.2 displacement from the RNA exit channel, facilitating promoter escape.
authors.affiliation: Department of Bacteriology, University of Wisconsin-Madison, 1550 Linden Drive, Madison, WI 53706, USA.
authors.forename: Jared T
authors.initials: JT
authors.lastname: Winkelman
authors.affiliation: Department of Bacteriology, University of Wisconsin-Madison, 1550 Linden Drive, Madison, WI 53706, USA.
authors.forename: Bradford T
authors.initials: BT
authors.lastname: Winkelman
authors.affiliation: Department of Bacteriology, University of Wisconsin-Madison, 1550 Linden Drive, Madison, WI 53706, USA.
authors.forename: Julian
authors.initials: J
authors.lastname: Boyce
authors.affiliation: Department of Bacteriology, University of Wisconsin-Madison, 1550 Linden Drive, Madison, WI 53706, USA.
authors.forename: Michael F
authors.initials: MF
authors.lastname: Maloney
authors.affiliation: Department of Bacteriology, University of Wisconsin-Madison, 1550 Linden Drive, Madison, WI 53706, USA.
authors.forename: Albert Y
authors.initials: AY
authors.lastname: Chen
authors.affiliation: Department of Bacteriology, University of Wisconsin-Madison, 1550 Linden Drive, Madison, WI 53706, USA.
authors.forename: Wilma
authors.initials: W
authors.lastname: Ross
authors.affiliation: Department of Bacteriology, University of Wisconsin-Madison, 1550 Linden Drive, Madison, WI 53706, USA. Electronic address: rgourse@bact.wisc.edu.
authors.forename: Richard L
authors.initials: RL
authors.lastname: Gourse
mesh_terms: D000596:Amino Acids / Q000737:chemistry; D001483:Base Sequence; D003432:Cross-Linking Reagents; D018360:Crystallography, X-Ray; D004269:DNA, Bacterial / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D011401:Promoter Regions, Genetic; D011487:Protein Conformation; D014158:Transcription, Genetic; D012407:rRNA Operon
chemical_list: D000596:Amino Acids; D003432:Cross-Linking Reagents; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D012321:DNA-Directed RNA Polymerases