Macromolecular complex formation is governed by two opposing constraints of specificity and speed.Kinetic and theoretical considerations suggest that significant rate enhancement can be achieved either by reducing the dimensionality of the search process or by the creation of a short-range attractive potential around the target site. This implies the existence of transient intermediates involving non-specific binding modes. We have shown that intermolecular paramagnetic relaxation enhancement (PRE) provides a means of directly detecting the presence and investigating the nature of low population transient intermediates under equilibrium conditions. Applying this approach, we have characterized the search process whereby a sequence-specific transcription factor (the homeodomain of Hox-D9) binds to non-cognate DNA sites as a means of enhancing the rate of specific association. The PRE data in the fast exchange regime reveal the presence of transient intermediates formed in a stochastic manner at non-cognate sites whose structure is similar to that of the specific complex. Two distinct search processes involving intra- as well as intermolecular translocations can be delineated. We have developed a novel approach for studying the kinetics of specific protein-DNA interactions by NMR exchange spectroscopy that involves the direct observation of translocation of a homeodomain between cognate sites on two oligonucleotide duplexes, differing by only a single base pair at the edge of the DNA recognition sequence. The single base pair change perturbs the 1H-15N correlation spectrum of a number of residues, while leaving the affinity for the DNA unchanged. The exchange process has apparent rate constants in the 5 to 20 s-1 range which are linearly dependent upon the concentration of free DNA. These rates are about three orders of magnitude larger than the dissociation rate constant determined by gel shift assays at nanomolar DNA concentrations. The complete NMR exchange data set, comprising auto- and cross-peak intensities as a function of mixing time at five concentrations of free DNA, can be fit simultaneously to a simple model in which protein translocation between DNA duplexes occurs via a second order process (with rate constants of 6x104 M-1s-1) involving direct collision of a protein-DNA complex with free DNA. This is akin to intersegmental transfer and a physical model for the process is discussed. Rapid translocation at high concentrations of free DNA observed directly by NMR exchange spectroscopy reconciles the long half-lives of protein-DNA complexes measured by biochemical analysis in vitro with the highly dynamic behavior of such complexes observed in vivo. Non-specific protein-DNA interactions are inherently dynamic involving both diffusion of the protein along the DNA and hoping of the protein from one DNA molecule or segment to another. Understanding how gene regulatory proteins interact non-specifically with DNA in terms of both structure and dynamics is challenging since the experimental observables are an ensemble average of many rapidly exchanging states. Using a variety of NMR spectroscopic techniques, including relaxation analysis, paramagnetic relaxation enhancement and residual dipolar couplings, we have characterized structural and kinetic aspects of the interaction of the HoxD9 homeodomain with a non-specific 24-bp DNA duplex in a system in which the protein is not constrained to any particular site. The data reveal that HoxD9 binds to non-specific DNA using the same binding mode and orientation as that observed in the specific complex. The mobility, however, of Arg side-chains contacting the DNA is increased in the non-specific complex relative to the specific one. The kinetics of intermolecular translocation between two different non-specific DNA molecules have also been analyzed and reveal that at high DNA concentrations (such as those present in vivo) direct transfer from one non-specific complex to another non-specific DNA molecule occurs without going through the intermediary of free protein. This provides a simple mechanism for accelerating the target search in vivo for the specific site in a sea of non-specific sites by permitting more effective sampling of available DNA sites as the protein jumps from one segment to another. We have also used 15Nz-exchange transverse relaxation optimized NMR spectroscopy to characterize the mechanistic details of intermolecular hopping for the multi-domain transcription factor, human Oct-1. Oct-1 is a member of the POU family of transcription factors and contains two helix-turn-helix DNA binding domains, POUHD and POUS, connected by a relatively short flexible linker. The two domains were found to exchange between specific sites at significantly different rates. The co-transcription factor, Sox2, decreases the exchange rate and equilibrium dissociation constant for Oct-1 ≥5-fold and 20-fold, respectively, by slowing the exchange rate for the POUS domain. DNA-dependent exchange rates measured at physiological ionic strength indicate that the two domains use both an intersegmental transfer mechanism, which does not involve the intermediary of free protein, and a fully dissociative or jumping mechanism to translocate between cognate sites. These data represent the first example dissecting domain-specific kinetics for protein-DNA association involving a multi-domain protein and provide evidence that intersegmental transfer involves a ternary intermediate or transition state in which the DNA-binding domains bridge two different DNA fragments simultaneously.
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