Conformation, dynamics, and structural transitions of the TATA box region of the self-complementary d[C-G)n-T-A-T-A-(C-G)n] duplexes in solution

Abstract

Structural and kinetic features of the TATA box located in the center of the alternating self-complementary d(C-G-C-G-T-A-T-A-C-G-C-G) duplex (TATA 12-mer) and d(C-G-C-G-C-G-T-A-T-A-C-G-C-G-C-G) duplex (TATA 16-mer) were probed by high-resolution proton and phosphorus NMR spectroscopy in aqueous solution. The imino exchangeable Watson-Crick protons and the nonexchangeable base protons in the TATA box of the TATA 12-mer and TATA 16 mer duplexes were assigned from intra and inter base pair nuclear Overhauser effect (NOE) measurements. Imino proton line-width and hydrogen exchange saturation recovery measurements deomonstrate that the dA.cntdot.dT base pairs in the TATA box located in the center of the TATA 12-mer and TATA 16-mer duplexes are kinetically more labile than flanking dG.cntdot.dC base pairs. The proton and phosphorus NMR parameters of the TATA 12-mer monitor a cooperative premelting transition in the TATA box prior to the onset of the melting transition to unstacked strands. Phosphorus NMR studies were unable to detect any indication of a right-handed B DNA to a left-handed Z DNA transition for the TATA 12-mer duplex in saturated NaCl solution. By contrast, the onset of the B to Z transition was detected for the TATA 16-mer in saturated NaCl solution. Proton and phosphorus NMR studies demonstrate formation of a loop conformation with chain reversal at the TATA segment for the TATA 12-mer and TATA 16-mer duplexes on lowering the DNA and counterion concentration. The imino protons (10-11 ppm) and phosphorus resonances (3.5-4.0 ppm; 4.5-5.0 ppm) of the loop segment fall in spectral windows well resolved from the corresponding markers in fully paired segments so that it should be possible to identify loops in longer DNA helixes. The equilibrium between the loop and fully paired duplex conformations of the TATA 12-mer and TATA 16-mer is shifted toward the latter on addition of moderate salt.