Ushiho Matsumoto

Characterization of the binding of HU and IHF, homologous histone-like proteins of Escherichia coli, to curved and uncurved DNA

The binding of E. coli histone-like protein HU to curved and uncurved DNA fragments containing adenine tracts was characterized by relative binding affinity assay, and compared with that of other homologous histone-like protein integration host factor (IHF). Both HU and IHF have about 3- to 5-fold higher affinity for overall curved DNA fragments such as (A6N4)11 and (A3T3N4)12 compared to a standard duplex fragment with mixed sequence. The binding manner of HU to the curved fragments was highly cooperative. However, loss of overall curvature for shorter fragments (< approximately 100 bp) reduced the preference of HU binding to curved (A3T3N4)n over uncurved (T3A3N4)n, indicating that the binding specificity of HU to curved DNA is length-dependent. Thus, the curved DNA configuration of the whole molecule facilitates the binding of several HU molecules to form the hierarchy of HU-DNA complex. Furthermore, it was shown that HU and IHF bind less well to (A6N9)n, which has a zig-zag straight structure, whereas they preferentially bind to uncurved (T3A3N4)14. These results suggested that not only intrinsically overall curvature but also the preferred orientations for DNA bending in the protein-DNA complex are important factors for affinities of HU and IHF.

Multiple conformations coexist and interchange in natural B DNA fibers

31P nuclear magnetic resonance (n.m.r.) of highly oriented NaDNA and LiDNA fibers was measured as a function of relative humidity over the range from 66% to 98%. The humidity dependence of the spectral patterns of NaDNA fibers shows that the A form has a single conformation while the B form has multiple conformations, and that interconversion between the A and B conformers in the transition region is slow compared to the n.m.r. time-scale (approximately 10(-5) s). Two major conformations of the B form of LiDNA are found to be stable at low relative humidity and they rapidly interchange at high relative humidities. The spectral patterns of immobilized LiDNA are compatible with the single-crystal structure of double-stranded oligo nucleic acids.

Specific and nonspecific interactions of integration host factor with oligo dnas as revealed by circular dichroism spectroscopy and filter binding assay

Binding specificity of integration host factor (IHF) to oligo DNAs has been studied by circular dichroism (CD) spectroscopy and filter binding experiment. CD difference spectra of IHF-DNA complexes demonstrated that a conformational change in DNA was induced by binding of IHF when DNA had a consensus sequence for the binding sites of IHF, but that such conformational change was not observed for consensus DNA 20 mer as well as nonconsensus DNA 45 mer. Dissociation constants for IHF-DNA complexes determined by filter binding assay showed that IHF has indeed stronger affinity to DNA with the consensus binding site than to nonconsensus DNA, but the difference in its affinity between consensus and nonconsensus DNAs was rather small, 3.4-fold. It was, therefore, concluded that the flanking regions of the consensus sequence are important for the specific binding of IHF and that its binding specificity is well characterized by the induced conformational change in DNA rather than by dissociation constants for IHF-DNA complexes.

Involvement of the histidine residues in the pH-induced conformational change of histone H5.

Comparative studies on the conformational stability of histones H1 and H5 have been carried out by monitoring the pH-induced conformational transitions of the proteins by CD and 1H NMR spectroscopies. The transition point of H1 agrees with the pKa of the carboxyl groups of the acidic residues. In contrast, the transition of H5 is associated with the ionization of the histidine residues which exist exclusively in H5, as well as the deionization of the acidic residues. These observations, combined with the result of the deuterium exchange rates of the histidine C-2 protons, led us to conclude that His-25 and His-62, which are buried in the globular domain, play an important role in the conformational stability of histone H5.