Our long-term goals is to understand the mechanism by which gene transcription in eukaryotes is initiated and regulated at the molecular level. The first step is to determine the mechanism by which the TATA binding protein (TBP) binds to specific sequences of DNA. The first step in the initiation of transcription is the binding of TBP to a promoter element 25 base pairs upstream from the initiation of transcription. The protein TFIIB binds next to form a ternary complex. Crystal structures are now available for several binary TBP-DNA complexes as well as for the ternary complex: TBP:DNA:TFIIB. Subsequently, other proteins are recruited until the pre- initiation TATA box. TBP binds to the minor groove of the DNA, widening the groove and producing two sharp kinks at the first and last steps of the TATA box where at each site a pair of phenylalannes is intercalated. The DNA is unwound within the box, and despite the overall 80 degree bind in the DNA, Watson-Crick base pairing is maintained. This unusual structural change in the DNA is associated with complex binding kinetics. Our approach to understanding the mechanism is to use rapid reaction kinetic techniques, combined with equilibrium measurements and a fluorescence detection method that can monitor the DNA bending and binding in real time. The approach involves varying the promoter sequence, the TBP, and solution conditions in order to map out the energetics and structural change along the reaction path. Experiments will test the hypothesis that intermediates in the TBP+DNA reaction play important roles in assuring that productive promoter sequences lead to proper and rapid assembly of the PIC. From an overall perspective it is clear that the regulation of gene transcription must be important in states of disease and of health and that understanding the mechanistic details of the regulation affords a rational approach to therapeutic design. A number of diseases, including many cancers, asthma, heart disease, and perhaps Alzheimer's disease are directly linked to altered synthesis of proteins and in a number of cases alterations in transcription have been demonstrated.
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