The TATA-box Binding Protein (TBP) is required by all three eukaryotic RNA
polymerases for the initiation of transcription from most promoters. TBP
recognizes, binds to, and bends promoter sequences called ``TATA-boxes'' in the
DNA. We present results from the study of individual Saccharomyces cerevisia
TBPs interacting with single DNA molecules containing a TATA-box. Using video
microscopy, we observed the Brownian motion of beads tethered by short
surface-bound DNA. When TBP binds to and bends the DNA, the conformation of the
DNA changes and the amplitude of Brownian motion of the tethered bead is
reduced compared to that of unbent DNA. We detected individual binding and
dissociation events and derived kinetic parameters for the process.
Dissociation was induced by increasing the salt concentration or by directly
pulling on the tethered bead using optical tweezers. In addition to the
well-defined free and bound classes of Brownian motion, we observed another two
classes of motion. These extra classes were identified with intermediate states
on a three-step, linear binding pathway. Biological implications of the
intermediate states are discussed.Comment: Accepted for publication in: Biophysical Journa