Single‐Molecule Characterization of Bacterial Factor‐Dependent Transcription Activation by Rob

ABSTRACT Factor‐dependent transcription activation is a key process of transcription regulation, involving complex interactions between multiple regulatory factors, RNA polymerase (RNAP), and promoter DNA, whose mechanism remains unclear. Here, we use single‐molecule magnetic trapping assays and fluorescence assays to characterize the kinetics of Escherichia coli ( E. coli ) RNAP transcription under the regulation of a pleiotropic AraC/XylS family factor, Rob, and elucidate their underlying mechanisms. We find that Rob prebinds RNAP holoenzyme to form a binary complex to facilitate promoter search. Upon promoter recognition, Rob enhances RNAP‐mediated promoter DNA unwinding to facilitate promoter escape and transition into transcription elongation. Rob activity is diversely regulated by small ligands, implying important roles of Rob C‐terminal domain (CTD) on its regulatory function. Our findings shed light on the mechanisms of Rob‐dependent transcription activation, which may provide insights into other transcription regulators in prokaryotes as well as eukaryotes.