Monitoring Structural Transitions in RNA Polymerase using Single Molecule FRET

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Structural transitions in protein machines play a central role in their function. Such motions can be studied in great detail and in real-time using the method of single-molecule FRET (smFRET); however, the use of smFRET in visualizing protein motions has been limited by a lack of strategies for site-specific labeling of mobile protein modules. To address this challenge, we have developed methods for site-specific introduction of fluorescent probes into proteins such as the bacterial RNA polymerase (RNAP); the methods are highly selective and combine unnatural-amino-acid mutagenesis and bio-orthogonal labeling. We combine this breakthrough with smFRET on surface-immobilized molecules and look at conformational change of a large structural domain in the RNAP, “β clamp”. For the first time, we show directly that the clamp is highly dynamic, visiting three conformations. We also show that some antibiotics that target RNAP function by locking clamp into specific conformations; further, we discovered that a major stress-response factor (ppGpp) functions by modulating clamp dynamics. We then employed a similar strategy to a more difficult target, a less than 45 amino acid motif, “trigger loop” (TL), which is very close to the active center of RNAP. We succeeded in producing a fully functional TL-labeled RNAP and used smFRET to show directly that binding of correct NTP at the active site induces TL closure and that the TL ensures substrate specificity. Finally, we show using a real-time assay that TL opens and closes in each nucleotide addition cycle. Our strategies are general and provide insight into functioning of large multi-subunit protein machines.