Single-molecule approach for studying RNAP II transcription initiation using magnetic tweezers

The initiation of transcription underlies the ability of cells to modulate genome expression as a function of both internal and external signals and the core process of initiation has features that are shared across all domains of life. Specifically, initiation can be sub-divided into promoter recognition, promoter opening, and promoter escape. However, the molecular players and mechanisms used are significantly different in Eukaryotes and Bacteria. In particular, bacterial initiation requires only the formation of RNA polymerase (RNAP) holoenzyme and proceeds as a series of spontaneous conformational changes while eukaryotic initiation requires the formation of the 31-subunit pre-initiation complex (PIC) and often requires ATP hydrolysis by the Ssl2/XPB subunit of the general transcription factor TFIIH. Our mechanistic view of this process in Eukaryotes has recently been improved through a combination of structural and single-molecule approaches which are providing a detailed picture of the structural dynamics that lead to the production of an elongation competent RNAP II and thus, an RNA transcript. Here we provide the methodological details of our single-molecule magnetic tweezers studies of transcription initiation using purified factors from Saccharomyces cerevisiae.