Base-pair opening dynamics measured by NMR: (1) 4-thio-thymidine incorporated DNA duplex. (2) The TATA binding protein-DNA complex

Melting temperatures and base pair opening rates were measured for normal DNA, 4-thio-thymidine incorporated DNA and PwTBP-TATA complex. Melting temperatures of d(GCTTATAAGC)2, d(GC4thioTTATAAGC)2, d(GCT4thioTATAAGC) 2, and d(GCTTA4thioTAAGC)2 were 33°C, 30°C, 25°C and 20°C, respectively. The chemical shift changes between d(GCTTATAAGC)2 and the other three 4-thio-thymidine incorporated DNA were smaller than 0.15 ppm, which was much smaller than that of 6-thio-guanine incorporated DNA. This result implies that the hydrogen bond between 4-thio-thymidine and adenine remains while the 6-thio-guanine and cytosine base pair lose its hydrogen bond. Base-pair lifetime times of 4-thio-dT:dA are 1--8 ms and they are 5--10 fold faster than dT:dA base pairs while the closing rates of the 4-thio-dT:dA and dT:dA are the same. Activation enthalpy and entropy of T4 in d(GCTTATAAGC)2 were 33.8 kcal/mol and 65.5 cal/mol/K while activation enthalpy and entropy of 4thioT4 in d(GCT4thioTATAAGC) 2 were 13.9 kcal/mol and 0.7 cal/mol/K. Equilibrium enthalpy and entropy of T4 in d(GCTTATAAGC)2 were 17.3 kcal/mol and 33.8 cal/mol/K while activation enthalpy and entropy of 4thioT4 in d(GCT4thioTATAAGC) 2 were 15.8 kcal/mol and 33.3 cal/mol/K.; Melting temperature of d(GCTTATAAGC)2 and PwTBP-TATA complex were 19°C and 49°C at pH 4.8. One dimensional spectra of TATA and PwTBP-TATA complex at 20°C showed that the imino peaks did not change at all upon binding protein to DNA, which indicates non-specific binding. When the temperature increased to 45°C, imino peaks were moved to new positions, which indicates that PwTBP binds DNA non-specifically at low temperature and the protein bends DNA with increase of temperature. Base-pair life times of C2 and T3 in PwTBP-TATA complex for C2 and T3 are 320 ms and 340 ms while those of T4 and T6 are 82 ms and 97 ms. This result showed a concerted structural fluctuations that opens a segment of DNA instead of individual base pair opening.