Distinct transcription initiation pathways of WT and P266L T7 RNAP.

(A) The transcription initiation pathways of WT T7 RNAP (top) and P266L T7 RNAP (bottom) are shown in cartoon format to illustrate the distinct intermediate conformations. The N-terminal domain is shown in blue, C terminal domain in red, DNA in black and RNA in green. Movement of the N terminal domain is marked by the arrow. Both WT and P266L T7 RNAP bind, bend, and open the promoter DNA from −4 to +2 to the same extent. The rigid C-linker of WT favors progressive rotation of the upstream end of the promoter to accommodate the growing hybrid from +4 to +6 positions, which pushes against the N-terminal domain driving the rotation of the promoter. The pushback from the N-terminal domain destabilizes the RNA:DNA hybrid and leads to abortive synthesis in WT. The flexible C-linker of P266L mutant (bottom panel) accommodates RNA extension up to 6 nt without significant promoter/N-terminal domain rotation. The reduced DNA scrunching together with the absence of promoter rotation in the 4–6 nt RNA range in P266L suggests that the growing hybrid is accommodated by an alternative pathway. After 6 nt RNA synthesis, promoter rotation and scrunching resumes in P266L. The weakened promoter interactions in WT after 9 nt RNA synthesis allow release of the N-terminal domain and transition into elongation. Persistent promoter interactions delay the transition in P266L. (B) Template strand scrunching in P266L RNAP with 7 bp RNA:DNA. Template strand from the IC3 (PDB: 1QLN) and IC7 (PDB: 3E2E) crystal structures showing decrease in the distance between C+1 and T-3 in the IC7 structure (brown) compared to the IC3 structure (blue).