Probingthe Rate-Limiting Step for IntramolecularTransfer of a Transcription Factor between Specific Sites on the SameDNA Molecule by 15Nz-ExchangeNMR Spectroscopy

摘要

The kinetics of translocation of the homeodomain transcription factor HoxD9 between specific sites of the same or opposite polarities on the same DNA molecule have been studied by ¹⁵Nz-exchange NMR spectroscopy. We show that exchange occurs by two facilitated diffusion mechanisms: a second-order intermolecular exchange reaction between specific sites located on different DNA molecules without the protein dissociating into free solution that predominates at high concentrations of free DNA, and a first-order intramolecular process involving direct transfer between specific sites located on the same DNA molecule. Control experiments using a mixture of two DNA molecules, each possessing only a single specific site, indicate that transfer between specific sites by full dissociation of HoxD9 into solution followed by reassociation is too slow to measure by z-exchange spectroscopy. Intramolecular transfer with comparable rate constants occurs between sites of the same and opposing polarity, indicating that both rotation-coupled sliding and hopping/flipping (analogous to geminate recombination) occur. Thehalf-life for intramolecular transfer (0.5–1 s) is many ordersof magnitude larger than the calculated transfer time (1–100μs) by sliding, leading us to conclude that the intramoleculartransfer rates measured by z-exchange spectroscopyrepresent the rate-limiting step for a one-base-pair shift from thespecific site to the immediately adjacent nonspecific site. At zeroconcentration of added salt, the intramolecular transfer rate constantsbetween sites of opposing polarity are smaller than those betweensites of the same polarity, suggesting that hopping/flipping may becomerate-limiting at very low salt concentrations.