The P266L mutation modifies both rotation and DNA scrunching changes during initiation.

(A) Cartoon illustration of FRET experiments to measure promoter rotation. The polymerase is in gray, the non-template strand in red and the template in green. Fluorescent donor TAMRA (red sphere) was introduced at position −22 in the non-template strand and acceptor Alexa 647 (blue square) at designated downstream positions on the template strand. Transcription complexes were walked to position N (+4 to +13) and FRET efficiency between donor (D) at −22 and acceptor (A) at N+5 was measured to obtain the D-A distances (R_DA, discontinuous line). (B and C) Average FRET efficiency and changes in D-A spatial distances are shown for P266L and WT T7 RNAP (D) Cartoon illustration of FRET experiments to measure DNA scrunching. The donor Cy3 (red sphere) was labeled on position −4 and acceptor Cy5 (blue square) labeled on downstream N+5 positions as above. (E and F) Average FRET efficiency and changes in averaged D-A spatial distances between Cy3 at the upstream edge (−4) and Cy5 at the downstream N+5 positions with the P266L and WT T7 RNAP complexes. The error bars of FRET efficiency represent the standard deviations from multiple independent measurements (N≥3).