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. 2009 Sep;37(17):5803-9.
doi: 10.1093/nar/gkp601. Epub 2009 Jul 20.

Nano Positioning System Reveals the Course of Upstream and Nontemplate DNA Within the RNA Polymerase II Elongation Complex

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Free PMC article

Nano Positioning System Reveals the Course of Upstream and Nontemplate DNA Within the RNA Polymerase II Elongation Complex

Joanna Andrecka et al. Nucleic Acids Res. .
Free PMC article

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 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.

Figures

Figure 1.
Figure 1.
Experimental design. (A) Labeling positions. Elongation complexes were formed using mismatched nucleic acid scaffolds. The template DNA strand, nontemplate DNA strand, and product RNA are colored blue, cyan, and red, respectively. Positions of attached dye molecules are indicated by green stars for donor and red stars for acceptor dye molecules. Bases whose positions were determined by crystallographic studies (6) are shown as solid circles. Those positions that could not be determined previously are shown as open circles. Pol II core (gray) and Rpb4/7 (red/blue) are shown schematically. A fluorescent dye molecule (Alexa 555 or TMR) that acted as the fluorescence donor in the smFRET experiments was attached to position +1, −2, −4, −7 −12, −15 or −18 of the nontemplate DNA. Since these are the positions that are to be determined the molecule is called the ‘antenna’. The acceptor dye molecule (Alexa 647) was used like a ‘satellite’ at a known position and was either attached to the template DNA (at positions −10, +3 or +9) or RNA (at −1, −4 or −10) or at one of two positions on the heterodimer Rpb4/7 (Rpb7-C150 and Rpb4-C73). (B) Overview of satellite positions. For each antenna position only those satellites whose distance to the antenna was expected to fall in the sensitive range for FRET measurements were used. Moreover, depending on the expected distance either Alexa 555 or TMR were used as fluorescence donor.
Figure 2.
Figure 2.
Position of nontemplate and upstream DNA in Pol II elongation complexes. (A) NPS results calculated from measured FRET values. Thirty-eight percent credibility volumes obtained for nontemplate DNA at register +1 (yellow), –2 (purple), –4 (orange), -7 (green), –12 (pink), –15 (dark salmon) and –18 (light blue) are displayed using surface representation. (Left) Side view of Pol II core enzyme shown in cartoon representation (gray), Rpb2 was omitted for clarity to reveal the nucleic acids. (Right) Alternate view of the polymerase (rotation by 90° as indicated). Here, all 10 polypeptides of the core enzyme are shown. The previously determined parts of the template DNA (dark blue), nontemplate DNA (light blue) and RNA (red) (6) are shown using surface representation. (B) Complete picture of the Pol II elongation complex. Obtained probability densities were used to build a model of nontemplate and upstream DNA. Modeled DNA is shown in cartoon representation. Both panels show the same orientations as in (A).
Figure 3.
Figure 3.
Consistency check of built model. A 38% credibility volume for dye molecule attached to nontemplate DNA at register –2 (purple) and –12 (pink) determined using NPS (surface representation) and computed accessible volume for a dye molecule attached to the respective position of the modeled nontemplate DNA (gray meshes).
Figure 4.
Figure 4.
Interaction network. Zoom into the elongation complex showing important structural features that define the path of the nontemplate DNA. Previously known DNA and RNA positions are shown in surface representation and modeled position of nontemplate and upstream DNA using cartoons. (A) Side view, polymerase elements important for the pathway of the nontemplate DNA are highlighted: rudder (green), fork loop1 (orange), fork loop 2 (yellow) and lobe loop (residues 272–278, pink). (B) A 90° rotated view (same orientation as Figure 2, right).

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