In bacteria, RNA polymerase (RNAP) binds to promoter DNA and unwinds ∼14 bp around the transcription start site to form a transcription bubble, with the unwound template (T) strand moving into the RNAP active center cleft. Transcription initiation is the most highly regulated step in gene expression. We summarized our work in a model for initial transcription, in which pausing is controlled by a complex set of determinants that modulate the transition from a 6- to a 7-nt RNA. We also obtained evidence for RNA backtracking during abortive initial transcription and for additional pausing prior to escape. Region sigma 3.2, which contains a loop blocking the RNA exit channel, was a major pausing determinant. Our work revealed a long pause (“initiation pause,” ∼20 s) after synthesis of a 6-mer RNA such pauses can serve as regulatory checkpoints.

Here, we studied initial transcription on a lac promoter using single-molecule fluorescence observations of DNA scrunching on immobilized transcription complexes. The mechanism of initial transcription is unclear due to the presence of transient intermediates and molecular heterogeneity. In bacteria, RNA polymerase (RNAP) initiates transcription by synthesizing short transcripts that are either released or extended to allow RNAP to escape from the promoter.

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