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Essential Components of Borreliella (Borrelia) burgdorferi In Vitro Transcription Assays
Published on: July 22, 2022
Single-Molecule Characterization of Bacterial Factor-Dependent Transcription Activation by Rob
Yuqiong Zhang1,2, Weijing Han2, Jing Shi3
1Institute of Fundamental and Frontier Sciences, University of Electronic Science and Technology of China, Chengdu, Sichuan, China.
None:
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.
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