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Biophys. J. BioFAST: First Published February 24, 2006. doi:10.1529/biophysj.105.074856
© 2006 by the Biophysical Society.

A more recent version of this article appeared on May 15, 2006.
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PROTEINS

Stepwise bending of DNA by a single TATA-box Binding Protein

Simon F Tolic-Norrelykke 1*, Mette B Rasmussen 2, Francesco S Pavone 3, Kirstine Berg-Sorensen 2 and Lene B Oddershede 2

1 Max Planck Institute for the Physics of Complex Systems
2 Niels Bohr Institute
3 University of Florence

* To whom correspondence should be addressed. E-mail: simonftn{at}pks.mpg.de.

Submitted on September 23, 2005
Revised on November 14, 2005
Accepted on 6 February 2006


  Abstract
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 cerevisiae 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.

Key Words: optical tweezers, single-molecule, tethered-particle-motion, transcription initiation, video microscopy




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Copyright © 2006 by the Biophysical Society.