NIRT: Watching Proteins Bend DNA with Subnanometer Resolution

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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.