Transcription is often regulated at the level of initiation by the presence of
transcription factors or nucleoid
proteins or by changing concentrations of metabolites. These can influence the kinetic properties and/or structures of the intermediate
RNA polymerase-
DNA complexes in the pathway. Time-resolved footprinting techniques combine the high temporal resolution of a stopped-flow apparatus with the specific structural information obtained by the probing agent. Combined with a careful quantitative analysis of the evolution of the signals, this approach allows for the identification and kinetic and structural characterization of the intermediates in the pathway of DNA sequence recognition by a
protein, such as a
transcription factor or
RNA polymerase. The combination of different probing agents is especially powerful in revealing different aspects of the conformational changes taking place at the
protein-
DNA interface. For example,
hydroxyl radical footprinting, owing to their small size, provides a map of the
solvent-accessible surface of the
DNA backbone at a single
nucleotide resolution; modification of the bases using
potassium permanganate can reveal the accessibility of the bases when the double helix is distorted or melted; cross-linking experiments report on the formation of specific
amino acid-
DNA contacts, and
DNase I footprinting results in a strong signal-to-noise ratio from
DNA protection at the binding site and
hypersensitivity at curved or
kinked DNA sites. Recent developments in
protein footprinting allow for the direct characterization of conformational changes of the
proteins in the complex.