Acceptor detected fluorescence resonance energy transfer for measurement up to 250 A and biophysical studies on core TATA binding protein-DNA complex.

摘要

We propose a general method to determine the molar absorptivity and natural lifetime of a dye attached to DNA based on the reaction of avidin and a biotinylated and dye labeled oligomer and report the spectroscopic properties of fluorescein, x-rhodamine, tetramethyl-rhodamine, attached to single strand and duplex DNA. The obtained spectroscopic parameters allowed us to develop an alternative approach enabling determination of distances to ∼250 A, and perhaps beyond, based on the time resolved acceptor detected FRET (trADFRET), using standard commercial fluorometric instrumentation and the common fluorescein-rhodamine dye pair. The trADFRET signals from double-labeled duplex DNA oligonucleotides show a rise phase due to pumping of the acceptor by the donor. A straightforward procedure eliminates the extraneous emissions from the donor and the directly excited acceptor dye to yield the pure sensitized emission of the acceptor. Measurements on the longest duplex gave an inter-dye distance of 201.5 A, a statistical error of +/- 1.3 A, and a difference from that predicted based on short oligonucleotides of 0.5 A. The technique allowed us to make solutions of "mock" oligonucleotides, which showed that with the dye pairs fluorescein: x-rhodamine, distances could be measured to 254 A with an error of +/- 6.7 A. Measurements on such long duplexes allow us to conclude that the effect on distance measurements of any departure of kappa 2 from the value 2/3 must be ∼1%. These results suggest that trADFRET could be used to detect conformational changes and assembly processes in very large macromolecular complexes exceeding 4MDa, such as the eukaryotic ribosomal translational initiation complex. We also studied the binding and bending of the AdMLP TATA sequence (TATAAAAG) by the core domain of yeast TBP (cTBP) allowing quantitation of the roles of the N-terminal domains (NTD) of yeast (yTBP) and human TBP (hTBP). The energetic patterns for hTBP and cTBP suggest that the 158aa NTD in hTBP does not initially occupy the DNA binding pocket. We find that the NTD's destabilize the three bound forms of DNA for both yTBP and hTBP. For all three proteins, the DNA bend angle (theta) depends on the TATA sequence, with theta for cTBP and hTBP > yTBP. For all three proteins, theta for the G6 variant (TATAAGAG) varies with temperature and increases in the presence of osmolyte to be similar to that of AdMLP. The results reported here reveal a clear structural difference for the bound DNA in solution vs. the crystal; we attribute the difference to the presence of osmolytes in the crystals.