| In RNAP holoenzyme and in the RNAP-promoter open complex, the sigmaR3/sigmaR4 linker is located within the RNA exit channel. The sigmaR3/sigmaR4 linker is unstructured and highly negatively charged (net charge = -8). It has been proposed that the sigmaR3/sigmaR4 linker serves as a "molecular mimic," or "molecular placeholder," for RNA in holoenzyme and open complex, and must be displaced to permit entry of RNA into the RNA exit channel during promoter escape---with displacement being driven by steric interactions with the RNA product when the RNA product reaches a length of ∼11 to ∼15 nt.; We used single-molecule fluorescence resonance energy transfer (FRET) to detect the structural changes that occur during the transition from initiation to elongation. We expanded existing two-color FRET method for three-component systems (three-color FRET). Three-color method allows simultaneous detection of three pair-wise distances to monitor multiple distance changes from a single experiment. By employing alternative laser excitation (ALEX) we defined stoichiometry ratios that allow virtual sorting of different populations in a multi-component system.; Using three-color ALEX we have directly detected displacement of the sigmaR3/sigmaR4 linker from the RNA exit channel, and have determined the dependence of sigmaR3/sigmaR4-linker displacement on RNA-product length.; The results establish that the sigmaR3/sigmaR4 linker is displaced during promoter escape. The results further establish that the sigmaR3/sigmaR4-linker is displaced in a single step upon synthesis of the RNA transcript 11 nt in length and that it remains at the same position for transcripts 12, 13, 14 and 15 nt in length. |
