Regulation of gene expression involves formation of specific protein-DNA complexes in which the DNA is often bent or sharply kinked. Kinetics measurements of DNA bending when in complex with the protein are essential for understanding the molecular mechanism that leads to precise recognition of specific DNAbinding sites. Previous kinetics measurements on several DNAbending proteins used stopped-flow techniques that have limited time resolution of few milliseconds. Here we use a nanosecond laser temperature-jump apparatus to probe, with submillisecond time resolution, the kinetics of bending͞unbending of a DNA substrate bound to integration host factor (IHF), an architectural protein from Escherichia coli. The kinetics are monitored with time-resolved FRET, with the DNA substrates end-labeled with a FRET pair. The temperature-jump measurements, in combination with stopped-flow measurements, demonstrate that the binding of IHF to its cognate DNA site involves an intermediate state with straight or, possibly, partially bent DNA. The DNA bending rates range from Ϸ2 ms ؊1 at Ϸ37°C to Ϸ40 ms ؊1 at Ϸ10°C and correspond to an activation energy of Ϸ14 ؎ 3 kcal͞mol. These rates and activation energy are similar to those of a single A:T base pair opening inside duplex DNA. Thus, our results suggest that spontaneous thermal disruption in base-paring, nucleated at an A:T site, may be sufficient to overcome the free energy barrier needed to partially bend͞kink DNA before forming a tight complex with IHF.DNA bending kinetics ͉ laser temperature jump ͉ protein-DNA interactions ͉ time-resolved FRET measurements

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The SWI/SNF chromatin remodeling complex changes the positions where nucleosomes are bound to DNA, exchanges out histone dimers, and disassembles nucleosomes. All of these activities depend on ATP hydrolysis by the catalytic subunit Snf2, containing a DNA-dependent ATPase domain. Here we examine the role of another domain in Snf2 called SnAC (Snf2 ATP coupling) that was shown previously to regulate the ATPase activity of SWI/SNF. We have found that SnAC has another function besides regulation of ATPase activity that is even more critical for nucleosome remodeling by SWI/SNF. We have found that deletion of the SnAC domain strongly uncouples ATP hydrolysis from nucleosome movement. Deletion of SnAC does not adversely affect the rate, processivity, or pulling force of SWI/SNF to translocate along free DNA in an ATP-dependent manner. The uncoupling of ATP hydrolysis from nucleosome movement is shown to be due to loss of SnAC binding to the histone surface of nucleosomes. While the SnAC domain targets both the ATPase domain and histones, the SnAC domain as a histone anchor plays a more critical role in remodeling because it is required to convert DNA translocation into nucleosome movement. The packing of DNA into chromatin involves the wrapping of 147 bp of DNA around a histone octamer to form a nucleosome and then assembly of nucleosomes into higher-order structures. Chromatin makes genomic DNA less accessible to protein factors important for transcription, replication, repair, and recombination. Chromatin structure is dynamic due to remodeling factors, some of which are ATP dependent, that facilitate making discrete regions accessible to other factors. ATP-dependent chromatin remodelers are single or large multisubunit assemblies composed of 1 to 17 subunits ranging from several kilodaltons to over a megadalton in molecular mass (1, 2). Each has a catalytic subunit with a conserved ATPase domain related to that of ATPdependent DNA helicases. In helicases, this domain couples ATP hydrolysis to DNA translocation and subsequent unwinding of double-stranded nucleic acid substrates by means of a translocase domain and a duplex destabilizing wedge domain (3-5). Unlike helicases, chromatin remodelers do not have nucleic acid unwinding activity, but have retained the translocase activity, which in turn repositions or disassembles nucleosomes (5, 6).Nucleosome movement by SWI/SNF-and ISWI-type complexes requires the ATPase domain to translocate along nucleosomal DNA near the dyad axis (7-10). DNA gaps near superhelical location 2 (SHL2) of the nucleosome block movement without interfering with binding of the remodeler. DNA translocation this far inside nucleosomes is challenging because there is no easy path for the ATPase domain to initially move. As the ATPase domain begins to translocate, it encounters histone-DNA interactions in both directions and has to overcome multiple histone-DNA interactions while trying to pull DNA into nucleosomes. The force required to disrupt histone-DNA interactions, as shown by mechanically ...

To elucidate the nature of the transition-state ensemble along the reaction pathway from a nonspecific protein-DNA complex to the specific complex, we have carried out measurements of DNA bending/unbending dynamics on a cognate DNA substrate in complex with integration host factor (IHF), an architectural protein from E. coli that bends its cognate site by ∼180°. We use a laser temperature jump to perturb the IHF-DNA complex and monitor the relaxation kinetics with time-resolved FRET measurements on DNA substrates end-labeled with a FRET pair. Previously, we showed that spontaneous bending/kinking of DNA, from thermal disruption of base-pairing/-stacking interactions, may be the rate-limiting step in the formation of the specific complex (

The dynamics and mechanism of how site-specific DNA-bending proteins initially interrogate potential binding sites prior to recognition have remained elusive for most systems. Here we present these dynamics for Integration Host factor (IHF), a nucleoid-associated architectural protein, using a μs-resolved T-jump approach. Our studies show two distinct DNA-bending steps during site recognition by IHF. While the faster (∼100 μs) step is unaffected by changes in DNA or protein sequence that alter affinity by >100-fold, the slower (1–10 ms) step is accelerated ∼5-fold when mismatches are introduced at DNA sites that are sharply kinked in the specific complex. The amplitudes of the fast phase increase when the specific complex is destabilized and decrease with increasing [salt], which increases specificity. Taken together, these results indicate that the fast phase is non-specific DNA bending while the slow phase, which responds only to changes in DNA flexibility at the kink sites, is specific DNA kinking during site recognition. Notably, the timescales for the fast phase overlap with one-dimensional diffusion times measured for several proteins on DNA, suggesting that these dynamics reflect partial DNA bending during interrogation of potential binding sites by IHF as it scans DNA.

Proteins that recognize and bind to specific sites on DNA often distort the DNA at these sites. The rates at which these DNA distortions occur are considered to be important in the ability of these proteins to discriminate between specific and nonspecific sites. These rates have proven difficult to measure for most protein-DNA complexes in part because of the difficulty in separating the kinetics of unimolecular conformational rearrangements (DNA bending and kinking) from the kinetics of bimolecular complex association and dissociation. A notable exception is the Integration Host Factor (IHF), a eubacterial architectural protein involved in chromosomal compaction and DNA recombination, which binds with subnanomolar affinity to specific DNA sites and bends them into sharp U-turns. The unimolecular DNA bending kinetics has been resolved using both stopped-flow and laser temperature-jump perturbation. Here we expand our investigation by presenting a global analysis of the ionic strength dependence of specific binding affinity and relaxation kinetics of an IHF-DNA complex. This analysis enables us to obtain each of the underlying elementary rates (DNA bending/unbending and protein-DNA association/dissociation), and their ionic strength dependence, even under conditions where the two processes are coupled. Our analysis indicates interesting differences in the ionic strength dependence of the bi- versus unimolecular steps. At moderate [KCl] (100-500 mM), nearly all the ionic strength dependence to the overall equilibrium binding affinity appears in the bimolecular association/dissociation of an initial, presumably weakly bent, encounter complex, with a slope SK(bi) ≈ 8 describing the loglog-dependence of the equilibrium constant to form this complex on [KCl]. In contrast, the unimolecular equilibrium constant to form the fully wrapped specific complex from the initial complex is nearly independent of [KCl], with SK(uni) < 0.5. This result is counterintuitive because there are at least twice as many ionic protein-DNA contacts in the fully wrapped complex than in the weakly bent intermediate. The following picture emerges from this analysis: in the bimolecular step, the observed [KCl]-dependence is consistent with the number of DNA counterions expected to be released when IHF binds nonspecifically to DNA whereas in the unimolecular reorganization step, the weak [KCl]-dependence suggests that two effects cancel one another. On one hand, formation of additional protein-DNA contacts in the fully wrapped complex releases bound counterions into bulk solution, which is entropically favored by decreasing [salt]. On the other hand, formation of the fully wrapped complex also releases tightly bound water molecules, which is osmotically favored by increasing [salt]. More generally, our global analysis strategy is applicable to other protein-DNA complexes, and opens up the possibility of measuring DNA bending rates in complexes where the unimolecular and bimolecular steps are not easily separable.

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RNA molecules are important factors involved in different cellular processes and have a multitude of roles in the cell. These roles include serving as a temporary copy of genes used for protein synthesis or functions in translational machinery. Interestingly, RNA is so far the only biological molecule that serves both as a catalyst (like proteins) and as information storage (like DNA). However, in contrast to proteins well known to be able to self-associate, such polymers have never been reported for natural RNA. We present here evidence that such a polymer of a natural RNA, the DsrA RNA, exists in the bacterial cell. DsrA is a small noncoding RNA (87 nucleotides) of Escherichia coli that acts by base-pairing to mRNAs in order to control their translation and turnover. One of the best-characterized target of DsrA is the rpoS mRNA. This messenger is of primary importance in the bacteria because it encodes the sigma S subunit of RNA polymerase and thus is a major regulator of stress response: the production of sigma S results in dramatic changes in cellular morphology and physiology. The results we obtained about DsrA self-assembly and our assumption about the function of this assembly in the cell will be presented herein.