Nano positioning system reveals the course of upstream and nontemplate DNA within the RNA polymerase II elongation complex.
Crystallographic studies of the RNA polymerase II (Pol II) elongation complex (EC) revealed the locations of downstream DNA and the DNA-RNA hybrid, but not the course of the nontemplate DNA strand in the transcription bubble and the upstream DNA duplex. Here we used single-molecule Fluorescence Resonance Energy Transfer (smFRET) experiments to locate nontemplate and upstream DNA with our recently developed Nano Positioning System (NPS). In the resulting complete model of the Pol II EC, separation of the nontemplate from the template strand at position +2 involves interaction with fork loop 2. The nontemplate strand passes loop beta10-beta11 on the Pol II lobe, and then turns to the other side of the cleft above the rudder. The upstream DNA duplex exits at an approximately right angle from the incoming downstream DNA, and emanates from the cleft between the protrusion and clamp. Comparison with published data suggests that the architecture of the complete EC is conserved from bacteria to eukaryotes and that upstream DNA is relocated during the initiation-elongation transition.
Andrecka
Joanna
J
Department of Chemistry and Biochemistry and Center for Integrated Protein Science München, Ludwig-Maximilians-Universität München, Butenandtstr.11, 81377 München, Germany.
Treutlein
Barbara
B
Arcusa
Maria Angeles Izquierdo
MA
Muschielok
Adam
A
Lewis
Robert
R
Cheung
Alan C M
AC
Cramer
Patrick
P
Michaelis
Jens
J
D001499:Bayes Theorem; D018360:Crystallography, X-Ray; D004247:DNA / Q000737:chemistry*; D031541:Fluorescence Resonance Energy Transfer; D008958:Models, Molecular; D012313:RNA / Q000737:chemistry; D012319:RNA Polymerase II / Q000737:chemistry*; D013698:Templates, Genetic; D014158:Transcription, Genetic
D012313:RNA; D004247:DNA; D012319:RNA Polymerase II