Fig. 2.

T7 RNAP transcription measured by single-molecule FRET. (A) Promoter DNA with Cy3 (donor) on the −4NT and Cy5 (acceptor) on +17T were surface-immobilized by using avidin-biotin linkage. Quartz surface was precoated with PEG (mixed with biotin-PEG) to achieve specific biotin conjugation and to prevent nonspecific interaction with the glass surface. (B) FRET histograms show E_corr on the x axis and counts of transcribing molecules on the y axis as T7 RNAP transcription was halted at designated positions by using limiting NTPs with or without 3′ dNTP. (C) Peak FRET efficiencies of R–P and halted transcribing complexes at various positions in IC (filled circles) and EC (open triangles) are shown (SEM came from more than two replicate measurements).