Fig. 1.

Pol II elongation complex. (A) Surface representation of a pol II elongation complex (PDB ID code 1Y1W) with structural elements highlighted: clamp in wheat, wall in violet, lid in green, rudder in yellow, fork loop 1 in orange, jaw in purple, and dock domain in aquamarine. Two putative RNA exit channels are indicated by red dash, labeled with 1 and 2. (B) The red star denotes a Cy5 on 10-nt RNA (GE2), situated next to the saddle. Green beads denote the Cy3 near the C terminus of Rpb4 in light pink or that of Rpb3 in cyan. The distance of Rpb4–GE2 is 82 Å and that of Rpb3–GE2 is 65 Å, respectively. (Inset) Cy3-CaM (orange) bound to CBP (light gray) extended from the C terminus of Rpb3 or Rpb4 in the presence of Ca²⁺ ions (yellow). (C) A design of triplet used for in-gel FRET. (Upper) Mixture of labeled and unlabeled pol II elongation complexes. Rpb3 or Rpb4 subunit is highlighted in pink; CaM is an orange dumbbell; Cy3 is green, and Cy5 is red. (Left) Pol II elongation complex with RNA unlabeled, a mixture of Cy3-CaM bound or unbound in the upper band, free Cy3-CaM in the lower band. (Middle) Pol II elongation complexes labeled with Cy5-RNA and Cy3-CaM. (Right) Same as Middle except unlabeled CaM is used. (D) Immobilized single molecules of pol II elongation complexes on a coated slide. (Left) Donor only, labeled with Cy3-CaM. (Middle) Donor and acceptor, with Cy3-CaM and Cy5-RNA. (Right) Acceptor only, with Cy5-RNA. A and B were prepared by PyMOL (50) (www.pymol.org).