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Studying transcription initiation by RNA polymerase with diffusion‐based single‐molecule fluorescence

Over the past decade, fluorescence‐based single‐molecule studies significantly contributed to characterizing the mechanism of RNA polymerase at different steps in transcription, especially in transcription initiation. Transcription by bacterial DNA‐dependent RNA polymerase is a multistep process that uses genomic DNA to synthesize complementary RNA molecules. Transcription initiation is a highly regulated step in E. coli, but it has been challenging to study its mechanism because of its stochasticity and complexity. In this review, we describe how single‐molecule approaches have contributed to our understanding of transcription and have uncovered mechanistic details that were not observed in conventional assays because of ensemble averaging.