Probing
the Rate-Limiting Step for Intramolecular
Transfer of a Transcription Factor between Specific Sites on the Same
DNA Molecule by 15Nz‑Exchange
NMR Spectroscopy
posted on 2015-12-17, 05:09authored byKyoung-Seok Ryu, Vitali Tugarinov, G. Marius Clore
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. The
half-life for intramolecular transfer (0.5–1 s) is many orders
of magnitude larger than the calculated transfer time (1–100
μs) by sliding, leading us to conclude that the intramolecular
transfer rates measured by z-exchange spectroscopy
represent the rate-limiting step for a one-base-pair shift from the
specific site to the immediately adjacent nonspecific site. At zero
concentration of added salt, the intramolecular transfer rate constants
between sites of opposing polarity are smaller than those between
sites of the same polarity, suggesting that hopping/flipping may become
rate-limiting at very low salt concentrations.