Mechanisms of specific and nonspecific binding of architectural proteins in prokaryotic gene regulation.
IHF and HU are small basic proteins of eubacteria that bind as homodimers to double-stranded DNA and bend the duplex to promote architectures required for gene regulation. These architectural proteins share a common alpha/beta fold but exhibit different nucleic acid binding surfaces and distinct functional roles. With respect to DNA-binding specificity, for example, IHF is sequence specific, while HU is not. We have employed Raman difference spectroscopy and gel mobility assays to characterize the molecular mechanisms underlying such differences in DNA recognition. Parallel studies of solution complexes of IHF and HU with the same DNA nonadecamer (5' --> 3' sequence: TC TAAGTAGTTGATTCATA, where the phage lambda H1 consensus sequence of IHF is underlined) show the following. (i) The structure of the targeted DNA site is altered much more dramatically by IHF than by HU binding. (ii) In the IHF complex, the structural perturbations encompass both the sugar-phosphate backbone and the bases of the consensus sequence, whereas only the DNA backbone is altered by HU binding. (iii) In the presence of excess protein, complexes of order higher than 1 dimer per duplex are detected for HU:DNA, though not for IHF:DNA. The results differentiate structural motifs of IHF:DNA and HU:DNA solution complexes, provide Raman signatures of prokaryotic sequence-specific and nonspecific recognition, and suggest that the architectural role of HU may involve the capability to recruit additional binding partners to even relatively short DNA sequences.
Benevides
James M
JM
School of Biological Sciences, University of MissouriKansas City, 5100 Rockhill Road, Kansas City, Missouri 64110-2499, USA.
Danahy
Jessica
J
Kawakami
Jessica
J
Thomas
George J
GJ
D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D004247:DNA / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D001411:Geobacillus stearothermophilus / Q000737:chemistry; D034982:Integration Host Factors / Q000737:chemistry / Q000378:metabolism*; D017433:Protein Structure, Secondary; D013059:Spectrum Analysis, Raman
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D034982:Integration Host Factors; C040330:histone-like protein HU, bacteria; C440502:integration host factor, E coli; D004247:DNA