Fluorescent methods to study transcription initiation and transition into elongation.

Abstract

The DNA-dependent RNA polymerases induce specific conformational changes in the promoter DNA during transcription initiation. Fluorescence spectroscopy sensitively monitors these DNA conformational changes in real time and at equilibrium providing powerful ways to estimate interactions in transcriptional complexes and to assess how transcription is regulated by the promoter DNA sequence, transcription factors, and small ligands. Ensemble fluorescence methods described here probe the individual steps of promoter binding, bending, opening, and transition into the elongation using T7 phage and mitochondrial transcriptional systems as examples.