NIRT: Watching Proteins Bend DNA with Subnanometer Resolution

Investigator(s):

Thomas Perkins (PI) ; Meredith Betterton (Co-PI) ; James Goodrich (Co-PI)

Sponsor:

University of Colorado at Boulder, CO 80309 3034926221

Start Date/Expiration Date

2004-08-01 to 2008-07-31 (amended 2004-07-20)

Awarded Amount to Date:

$1,490,000

Abstract: This proposal was received in response to Nanoscale Science and Engineering initiative, NSF 03-043, category NIRT. This interdisciplinary research effort will combine high-resolution single-molecule experiments and mathematical modeling with ensemble biochemical assays to study protein-DNA interactions that control gene expression. The human transcription factor TATA box-binding protein (TBP) is the central protein in a larger protein complex (TFIID) that binds to DNA sequences in genes and controls how genes are transcribed into messenger RNA. The proposed project will investigate the mechanism of DNA binding by single TBP molecules and TFIID complexes to illuminate a key first step in transcription initiation. A unique optical trapping instrument with sub-nanometer resolution will be developed to enable single-molecule studies of biological complexes. The improved apparatus will allow direct detection of the binding of TBP to TATA-box sequences, via the apparent shortening of a DNA molecule due to the 100-degree kink in the DNA backbone caused by TBP. Ensemble biochemical experiments will be carefully coordinated for comparison with the innovative single-molecule results. Theoretical analysis of the experimental data will determine the bend angle, and provide testable predictions for the search time required by TBP to find the TATA box. The analysis will enable an energetic description of the binding as a function of DNA tension and will further illuminate the structure of multi-protein complexes bound to DNA, by predicting the different experimental signatures of protein-induced bending of the DNA and wrapping of the DNA around the complex. Experiments and theoretical analysis will examine how DNA length, the number of TATA-box sequences per DNA construct, and the presence of other DNA-binding obstacle proteins affect the time for TBP to find a TATA box. The project takes a multifaceted approach to education that will include training of undergraduate and graduate students in research, mentoring students in course development and science teaching, and using a unique mechanism to explain the science to the general public. Students will develop a deep understanding of biophysics and its interrelation to other disciplines. Joint meetings of the interdisciplinary research team will bring together students and researchers with backgrounds including applied math, biology, and physics.

NSF Org:

PHY - Division of Physics

Award Number:

0404286

Award Instrument:

Standard Grant

Program Manager:

C. Denise Caldwell
PHY Division of Physics
MPS Directorate for Mathematical & Physical Sciences

NSF Program(s):

BIOCHEMICAL & BIOMASS ENG, BIOLOGICAL PHYSICS, NANOSCALE: INTRDISCPL RESRCH T

Field Application(s):

Other nsf.applications NEC

Program Reference Code(s):

BIOCHEMICAL & BIOMASS ENG, 1402
BIOLOGICAL PHYSICS, 7246
Nanoscale Research-for PHY use only, 1767
NANOSCALE: INTRDISCPL RESRCH T, 1674
UNASSIGNED, 0000

Program Element Code(s):

1402
BIOLOGICAL PHYSICS, 7246
NANOSCALE: INTRDISCPL RESRCH T, 1674