Résumé
Abstract Live cell RNA imaging revealed that transcription levels are encoded by the intrinsic dynamics of promoters. However, capturing both kinetic and molecular aspects of promoter fluctuations has been challenging. Here, we resolve this key issue by combining Single Molecule DNA footprinting (SMF) with live transcription imaging. Using HIV-1 as a model, SMF reveals that the promoter functions in two modes depending on the viral transactivator Tat. Without Tat, a nucleosome occupies the core promoter and prevents assembly of the pre-initiation complex. With Tat, this nucleosome is absent while TBP and initiating polymerases are frequently detected. Combining live imaging with SMF provides a mechanistic model of promoter dynamics, which estimates the rates of deposition and removal of promoter nucleosomes (0.7 h - 1 ), TBP binding (0.04 min - 1 ) and polymerase loading (seconds). The data further reveal a kinetic proofreading mechanism of initiating polymerases, which enables Tat to indirectly control promoter nucleosomes by promoting elongation.