Nano positioning system reveals the course of upstream and nontemplate DNA within the RNA polymerase II elongation complex

Abstract

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 β10-β11 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.

Single molecule studies of RNA polymerase II transcription in vitro

“…The RNAP II elongation complex has also been investigated using a NPS to monitor positional fluctuations of upstream DNA and the non-template DNA strand. 14 The angle between the upstream and downstream duplexes was found to be ~80°, consistent with structural data from bacterial polymerases. 17 Upstream DNA from -5 to -10 was exposed, while the template strand downstream of -5 was in the RNAP II cleft.…”

T7 RNA Polymerases Backed up by Covalently Trapped Proteins Catalyze Highly Error Prone Transcription

“…RNAPs open the downstream DNA duplex at the DNA entry site to generate a transcription bubble, in which the transcribed strand (TS) is delivered deep into the active site and used for nascent RNA synthesis, whereas the nontranscribed strand (NTS) is relatively exposed to the surface of RNAP (17)(18)(19). The resolution of the crystal structure of yeast RNAP II revealed that conventional bulky lesions such as a cyclobutane pyrimidine dimer, a cisplatin intrastrand cross-link, and a monofunctional platinum adduct in the TS are delivered to the active site or its proximal position and then arrest transcription (20 -22).…”

Molecular Mechanisms of Transcription Initiation—Structure, Function, and Evolution of TFE/TFIIE-Like Factors and Open Complex Formation

“…This model is based on distance constraints derived from smFRET measurements between fluorescent dye pairs introduced at strategic locations in components of the OC [38]. Interprobe distances were calculated from smFRET measurements and processed using the NPS system [86]. …”