8 research outputs found
Pressure dissociation of integration host factorāDNA complexes reveals flexibility-dependent structural variation at the proteināDNA interface
E. coli Integration host factor (IHF) condenses the bacterial nucleoid by wrapping DNA. Previously, we showed that DNA flexibility compensates for structural characteristics of the four consensus recognition elements associated with specific binding (Aeling et al., J. Biol. Chem. 281, 39236ā39248, 2006). If elements are missing, high-affinity binding occurs only if DNA deformation energy is low. In contrast, if all elements are present, net binding energy is unaffected by deformation energy. We tested two hypotheses for this observation: in complexes containing all elements, (1) stiff DNA sequences are less bent upon binding IHF than flexible ones; or (2) DNA sequences with differing flexibility have interactions with IHF that compensate for unfavorable deformation energy. Time-resolved Fƶrster resonance energy transfer (FRET) shows that global topologies are indistinguishable for three complexes with oligonucleotides of different flexibility. However, pressure perturbation shows that the volume change upon binding is smaller with increasing flexibility. We interpret these results in the context of Record and coworker's model for IHF binding (J. Mol. Biol. 310, 379ā401, 2001). We propose that the volume changes reflect differences in hydration that arise from structural variation at IHFāDNA interfaces while the resulting energetic compensation maintains the same net binding energy
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Pressure dissociation of integration host factor-DNA complexes reveals flexibility-dependent structural variation at the protein-DNA interface
E. coli Integration host factor (IHF) condenses the bacterial nucleoid by wrapping DNA. Previously, we showed that DNA flexibility compensates for structural characteristics of the four consensus recognition elements associated with specific binding (Aeling et al., J. Biol. Chem. 281, 39236ā39248, 2006). If elements are missing, high-affinity binding occurs only if DNA deformation energy is low. In contrast, if all elements are present, net binding energy is unaffected by deformation energy. We tested two hypotheses for this observation: in complexes containing all elements, (1) stiff DNA sequences are less bent upon binding IHF than flexible ones; or (2) DNA sequences with differing flexibility have interactions with IHF that compensate for unfavorable deformation energy. Time-resolved Fƶrster resonance energy transfer (FRET) shows that global topologies are indistinguishable for three complexes with oligonucleotides of different flexibility. However, pressure perturbation shows that the volume change upon binding is smaller with increasing flexibility. We interpret these results in the context of Record and coworker's model for IHF binding (J. Mol. Biol. 310, 379ā401, 2001). We propose that the volume changes reflect differences in hydration that arise from structural variation at IHFāDNA interfaces while the resulting energetic compensation maintains the same net binding energy
A vector set for systematic metabolic engineering in Saccharomyces cerevisiae.
A set of shuttle vectors was constructed to facilitate expression of genes for metabolic engineering in Saccharomyces cerevisiae. Selectable markers include the URA3, TRP1, MET15, LEU2-d8, HIS3 and CAN1 genes. Differential expression of genes can be achieved as each marker is available on both CEN/ARS- and 2 Āµ-containing plasmids. Unique restriction sites downstream of TEF1, PGK1 or HXT7-391 promoters and upstream of the CYC1 terminator allow insertion of open-reading frame cassettes for expression. Furthermore, a fragment appropriate for integration into the genome via homologous recombination can be readily generated in a polymerase chain reaction. Vector marker genes are flanked by loxP recognition sites for the CreA recombinase to allow efficient site-specific marker deletion and recycling. Expression and copy number were characterized for representative high- and low-copy vectors carrying the different marker and promoter sequences. Metabolic engineering typically requires the stable introduction of multiple genes and genomic integration is often preferred. This requires an expanded number of stable expression sites relative to standard gene expression studies. This study demonstrated the practicality of polymerase chain reaction amplification of an expression cassette and genetic marker, and subsequent replacement of endogenous retrotransposons by homologous recombination with flanking sequences. Such reporters were expressed comparably to those inserted at standard integration loci. This expands the number of available characterized integration sites and demonstrates that such sites provide a virtually inexhaustible pool of integration targets for stable expression of multiple genes. Together these vectors and expression loci will facilitate combinatorial gene expression for metabolic engineering
Electrophoretic mobility-shift assay of IHF binding to oligonucleotide A
<p><b>Copyright information:</b></p><p>Taken from "Pressure dissociation of integration host factorāDNA complexes reveals flexibility-dependent structural variation at the proteināDNA interface"</p><p></p><p>Nucleic Acids Research 2007;35(6):1761-1772.</p><p>Published online 25 Feb 2007</p><p>PMCID:PMC1874591.</p><p>Ā© 2007 The Author(s)</p>2. IHF concentrations in Lanes 1ā10 are 0, 20, 40, 60, 81, 99, 120, 165, 201 and 240ānM, respectively. This pseudo-color image was generated by coloring the emission collected through a 520-nm band pass filter green (FAM fluorescence) and coloring the emission collected through a 580-nm band pass filter red (TAMRA fluorescence). With excitation at 488ānm, the unliganded oligonucleotide is green, reflecting only FAM fluorescence. The yellow color of the mobility-shifted band results from a combination of green and red fluorescence, indicating efficient FRET due to the wrapped DNA in the bound complex
Panel A shows the pressure FRET ratio baseline data (open circle) and polynomial smoothing curve (solid line for oligonucleotide A
<p><b>Copyright information:</b></p><p>Taken from "Pressure dissociation of integration host factorāDNA complexes reveals flexibility-dependent structural variation at the proteināDNA interface"</p><p></p><p>Nucleic Acids Research 2007;35(6):1761-1772.</p><p>Published online 25 Feb 2007</p><p>PMCID:PMC1874591.</p><p>Ā© 2007 The Author(s)</p>6 in the absence of IHF compared with unprocessed data for 10ānM DNA and 25ānM IHF (filled square) (10āmM Tris pHā8.0, 100āmM NaCl and 1āmM EDTA). Panel B compares fraction bound for oligonucleotides A.2 (filled diamond) and A.6 (filled square) at 10ānM DNA, 25ānM IHF, i.e. same A.6 data as panel A and same reaction conditions. Solid and dashed curves are the fits and 95% confidence intervals to these individual experiments, using equations () as described in the text