Conformational Heterogeneity in RNA Polymerase Observed by Single-Pair FRET Microscopy

Kinetic, structural, and single-molecule transcription measurements suggest that RNA polymerase can adopt many different conformations during elongation. We have measured the geometry of the DNA and RNA in ternary elongation complexes using single-pair fluorescence resonance energy transfer. Six different synthetic transcription elongation complexes were constructed from DNA containing an artificial transcription bubble, an RNA primer, and core RNA polymerase from Escherichia coli. Two different RNA primers were used, an 8-mer and a 5′-extended 11-mer. Fluorescent dye labels were attached at one of three positions on the DNA and at the RNA primer 5′-end. Structurally, the upstream DNA runs perpendicular to the proposed RNA exit channel. Upon nucleoside-triphosphate addition, DNA/RNA hybrid separation occurs readily in the 11-mer complexes but not in the 8-mer complexes. Clear evidence was obtained that RNA polymerase exists in multiple conformations among which it fluctuates.