Comparison of protein binding to DNA in vivo and in vitro: defining an effective intracellular target.

Yang, Shu-Wei; Nash, Howard A.

doi:10.1002/j.1460-2075.1995.tb00319.x

Cited by 102 publications

(115 citation statements)

References 41 publications

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“…Single-molecule techniques allowed us to tackle this question in quantitative terms by using a DNA macromolecule and shed light on the contribution of IHF to the large-scale structure of the bacterial nucleoid. Our conclusions underscore the importance of IHF low-affinity binding, in agreement with other studies (25,30). Similar studies of other histone-like proteins, being carried out now, may allow for a comparison of the relative contribution of these proteins in shaping the bacterial nucleoid.…”

Section: Number Of Bound Ihf Molecules In a -Dna-ihf Complexsupporting

confidence: 91%

“…The numbers are even higher in stationary phase, the concentration increasing 5-to 6-fold. This high concentration of IHF, for a protein having a limited number of high-affinity sites, suggests that nonspecific binding has an important biological role (30). Recent in vivo measurements suggest that the vast majority of IHF molecules, about 10 M in growing cells, are found bound to DNA (26,30).…”

Section: Number Of Bound Ihf Molecules In a -Dna-ihf Complexmentioning

confidence: 99%

“…This high concentration of IHF, for a protein having a limited number of high-affinity sites, suggests that nonspecific binding has an important biological role (30). Recent in vivo measurements suggest that the vast majority of IHF molecules, about 10 M in growing cells, are found bound to DNA (26,30). It is interesting to note that the ratio of the number of base pairs in an E. coli genome (4.7 ϫ 10 6 bp), to the number of IHF molecules in a cell in exponential phase is about the same as in our experiments, at IHF concentrations at which further addition of IHF does not bring forth further compaction.…”

Section: Number Of Bound Ihf Molecules In a -Dna-ihf Complexmentioning

confidence: 99%

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Compaction of single DNA molecules induced by binding of integration host factor (IHF)

Ali

Amit

Braslavsky

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

116

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We studied the interaction between the integration host factor (IHF), a major nucleoid-associated protein in bacteria, and single DNA molecules. Force-extension measurements of DNA and an analysis of the Brownian motion of small beads tethered to a surface by single short DNA molecules, in equilibrium with an IHF solution, indicate that: (i) the DNA-IHF complex retains a random, although more compact, coiled configuration for zero or small values of the tension, (ii) IHF induces DNA compaction by binding to multiple DNA sites with low specificity, and (iii) with increasing tension on the DNA, the elastic properties of bare DNA are recovered. This behavior is consistent with the predictions of a statistical mechanical model describing how proteins bending DNA are driven off by an applied tension on the DNA molecule. Estimates of the amount of bound IHF in DNA-IHF complexes obtained from the model agree very well with independent measurements of this quantity obtained from the analysis of DNA-IHF crosslinking. Our findings support the long-held view that IHF and other histone-like proteins play an important role in shaping the longscale structure of the bacterial nucleoid.T he genetic material in bacterial cells is organized in a structure called the nucleoid (1-3). In Escherichia coli, this nucleoprotein complex consists of a single circular DNA molecule 4.7 million bp long, RNA, and a large variety of bound proteins. Among these, about 10 so-called histone-like proteins, including HU, integration host factor (IHF), and H-NS (1-3), shape the short-scale structure of the nucleoid by bending DNA locally on binding. These proteins therefore play an important role in compacting the DNA molecule, in addition to other factors such as supercoiling (4), macromolecular crowding (5), and osmotic effects (6).The level of nucleoid-associated proteins changes as a function of bacterial growth. For example, the level of IHF was shown to increase on entry to the stationary phase of growth, becoming one of the major histone-like proteins in the cell (7-10). By binding to specific DNA sites, IHF participates in forming higher-order DNA structures required for replication, sitespecific recombination, phage packaging, and regulation of transcription initiation (8). IHF can also bind to DNA nonspecifically and can be substituted by HU. In fact, IHF and HU possess similar overall structures and share several regions of conserved homologies (11,12).Very little is known about the structural modifications on DNA induced by histone-like proteins in nucleoid formation. In particular, the degree of compaction induced by each of these proteins has not been quantified, and information about the large-scale structure of nucleoprotein complexes is scarce. This deficiency stems in part from the fact that classical techniques used in molecular biology are designed for studying relatively strong DNA-binding sites and cannot appropriately assess the contribution of nonspecific low-affinity interactions. Furthermore, analyses based on electron micr...

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Section: Number Of Bound Ihf Molecules In a -Dna-ihf Complexsupporting

confidence: 91%

Section: Number Of Bound Ihf Molecules In a -Dna-ihf Complexmentioning

confidence: 99%

Section: Number Of Bound Ihf Molecules In a -Dna-ihf Complexmentioning

confidence: 99%

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Compaction of single DNA molecules induced by binding of integration host factor (IHF)

Ali

Amit

Braslavsky

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

116

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“…However, unlike the HU proteins, IHF has evolved a sequence-dependent, high affinity, DNA-binding motif. The total intracellular concentration of IHF is maintained at a level of greater than 1 M, but its free concentration is estimated to be between 0.7 and 5 nM (14,46). Therefore, because the K d value for IHF binding to its highest affinity, sequence-dependent DNA-binding sites is about 1 ϫ 10 Ϫ9M , these high affinity sites are occupied most of the time and function as constitutive architectural nucleoprotein structures on the bacterial chromosome.…”

Section: Discussionmentioning

confidence: 99%

Indirect Recognition in Sequence-specific DNA Binding by Escherichia coli Integration Host Factor

Aeling

Opel

Steffen

et al. 2006

Journal of Biological Chemistry

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Integration host factor (IHF) is a bacterial histone-like protein whose primary biological role is to condense the bacterial nucleoid and to constrain DNA supercoils. It does so by binding in a sequence-independent manner throughout the genome. However, unlike other structurally related bacterial histone-like proteins, IHF has evolved a sequence-dependent, high affinity DNA-binding motif. The high affinity binding sites are important for the regulation of a wide range of cellular processes. A remarkable feature of IHF is that it employs an indirect readout mechanism to bind and wrap DNA at both the nonspecific and high affinity (sequence-dependent) DNA sites. In this study we assessed the contributions of pre-formed and protein-induced DNA conformations to the energetics of IHF binding. Binding energies determined experimentally were compared with energies predicted for the IHF-induced deformation of the DNA helix (DNA deformation energy) in the IHF-DNA complex. Combinatorial sets of de novo DNA sequences were designed to systematically evaluate the influence of sequence-dependent structural characteristics of the conserved IHF recognition elements of the consensus DNA sequence. We show that IHF recognizes pre-formed conformational characteristics of the consensus DNA sequence at high affinity sites, whereas at all other sites relative affinity is determined by the deformational energy required for nearest-neighbor base pairs to adopt the DNA structure of the bound DNA-IHF complex.Site-specific DNA binding by regulatory proteins is a feature of the regulatory processes that maintain, expand, and express genetic information such as replication, recombination, transposition, and transcription. The chemical and physical mechanisms that underlie sequence-specific recognition of regulatory elements by cognate DNA-binding proteins are typically classified as direct versus indirect readout. The former refers primarily to hydrogen bonds between proteins and the unique extra-cyclic substituents at C-4 of pyrimidines, C-6 of purines, and N-7 of purines. These groups provide a base pair-specific pattern of hydrogen bond donors and acceptors in the major groove of DNA that can be directly read by a complementary pattern of amino acid side chain donors and acceptors. Indirect readout refers to recognition of aspects of DNA structure such as intrinsic curvature, topology of major and minor grooves, ordered water structures, local geometry of backbone phosphates, and flexibility or deformability. Because both the local DNA structure and energy to deform DNA are themselves intrinsic sequence-dependent properties, the conserved sequences that distinguish binding sites necessarily include contributions from both direct and indirect mechanisms. Consequently, although the contribution from indirect mechanisms is expected to be significant in protein-DNA complexes that feature substantial DNA deformation, it has proven difficult to evaluate these contributions quantitatively. A protein that relies exclusively, or primarily, on indir...

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“…IHF has also been shown to function as a DNA looper protein to facilitate interactions between regulatory proteins bound at upstream sites and RNA polymerase at downstream promoter sites (15,16). Because these functions involve IHF binding to site-specific high affinity sites and because of the high intracellular concentration of this abundant chromosomal organizer protein, these IHF sites are likely saturated under all physiological conditions (17). Thus, unlike other regulatory proteins that bind small molecule effectors that affect their DNA binding properties, IHF functions as an architectural component of DNA structures that affect the constitutive or basal level expression of many promoters.…”

mentioning

confidence: 99%

Global Gene Expression Profiling in Escherichia coliK12

Arfin

Long

Ito

et al. 2000

Journal of Biological Chemistry

253

202

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We have used nylon membranes spotted in duplicate with full-length polymerase chain reaction-generated products of each of the 4,290 predicted Escherichia coli K12 open reading frames (ORFs) to measure the gene expression profiles in otherwise isogenic integration host factor IHF ؉ and IHF ؊ strains. Our results demonstrate that random hexamer rather than 3 ORF-specific priming of cDNA probe synthesis is required for accurate measurement of gene expression levels in bacteria. This is explained by the fact that the currently available set of 4,290 unique 3 ORF-specific primers do not hybridize to each ORF with equal efficiency and by the fact that widely differing degradation rates (steady-state levels) are observed for the 25-base pair region of each message complementary to each ORF-specific primer. To evaluate the DNA microarray data reported here, we used a linear analysis of variance (ANOVA) model appropriate for our experimental design. These statistical methods allowed us to identify and appropriately correct for experimental variables that affect the reproducibility and accuracy of DNA microarray measurements and allowed us to determine the statistical significance of gene expression differences between our IHF ؉ and IHF ؊ strains. Our results demonstrate that small differences in gene expression levels can be accurately measured and that the significance of differential gene expression measurements cannot be assessed simply by the magnitude of the fold difference. Our statistical criteria, supported by excellent agreement between previously determined effects of IHF on gene expression and the results reported here, have allowed us to identify new genes regulated by IHF with a high degree of confidence.

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Comparison of protein binding to DNA in vivo and in vitro: defining an effective intracellular target.

Cited by 102 publications

References 41 publications

Compaction of single DNA molecules induced by binding of integration host factor (IHF)

Compaction of single DNA molecules induced by binding of integration host factor (IHF)

Indirect Recognition in Sequence-specific DNA Binding by Escherichia coli Integration Host Factor

Global Gene Expression Profiling in Escherichia coliK12

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