Oligomerization of the chromatin‐structuring protein H‐NS

Smyth, Clare P.; Lundbäck, Thomas; Renzoni, Debora; Siligardi, Giuliano; Beavil, Rebecca L.; Layton, Meredith J.; Sidebotham, Julie M.; Hinton, Jay C. D.; Driscoll, Paul C.; Higgins, Christopher F.; Ladbury, John E.

doi:10.1046/j.1365-2958.2000.01917.x

Cited by 111 publications

(120 citation statements)

References 32 publications

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“…The presence of a species sedimenting at 1.8 S then reveals abnormal sedimentation behavior for the wild-type pro- tein, since this value is smaller than the one obtained for the ⌬64 dimer. It must be concluded, in good agreement with recent NMR experiments (38), that the shape of the H-NS wild type is far from spherical. Indeed, the NMR signal of the whole H-NS protein was found to be strikingly similar to that of its amino-terminal domain, suggesting that the latter is moving freely in the whole protein.…”

Section: Oligomerization State Of the Various H-ns Proteins In Solutisupporting

confidence: 86%

“…Oligomerization of this core occurs in solution as well as when the protein is stably bound to DNA. All of the various NH 2 -terminal domains of H-NS-like proteins examined to date are reported to be dimeric 3 with the notable exception of the ⌬64 peptide from S. typhimurium (38). E. coli ⌬64 H-NS analyzed here by equilibrium sedimentation behaves as a dimer.…”

Section: Discussionmentioning

confidence: 74%

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The Degree of Oligomerization of the H-NS Nucleoid Structuring Protein Is Related to Specific Binding to DNA

Badaut

Williams

Arluison

et al. 2002

Journal of Biological Chemistry

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At several E. coli promoters, initiation of transcription is repressed by a tight nucleoprotein complex formed by the assembly of the H-NS protein. In order to characterize the relationship between the structure of H-NS oligomers in solution and on relevant DNA fragments, we have compared wild-type H-NS and several transdominant H-NS mutants using gel shift assays, DNase I footprinting, analytical ultracentrifugation, and reactivity toward a cross-linking reagent. In solution, oligomerization occurs through two protein interfaces, one necessary to construct a dimeric core (and involving residues 1-64) and the other required for subsequent assembly of these dimers. We show that, as well as region 64 -95, residues present in the NH 2 -terminal coiled coil domain also participate in this second interface. Our results support the view that the same interacting interfaces are also involved on the DNA. We propose that the dimeric core recognizes specific motifs, with the second interface being critical for their correct head to tail assembly. The COOH-terminal domain of the protein contains the DNA binding motif essential for the discrimination of this specific functional assembly over competitive nonspecific H-NS polymers.

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Section: Oligomerization State Of the Various H-ns Proteins In Solutisupporting

confidence: 86%

Section: Discussionmentioning

confidence: 74%

The Degree of Oligomerization of the H-NS Nucleoid Structuring Protein Is Related to Specific Binding to DNA

Badaut

Williams

Arluison

et al. 2002

Journal of Biological Chemistry

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“…We also observed the more dispersed phenotype with immunofluorescence imaging of H-NS, but did not present this comparison here because fixation of the bacterial nucleoid is in general discouraged. It is known that H-NS oligomerizes/clusters even without fusion to fluorescent proteins (28,29), and this intrinsic H-NS clustering effect is important for its in vivo function as a transcriptional silencer (29) and brings gene loci in different chromosome regions into spatial proximity in wild-type cells (27). However, fusion with mEos2, PAmCherry, mMaple, and tdEos may have exaggerated the H-NS clustering effect.…”

Section: Dimerization Tendency Of Photoactivatable Fluorescent Proteinsmentioning

confidence: 99%

Characterization and development of photoactivatable fluorescent proteins for single-molecule–based superresolution imaging

Wang¹,

Moffitt²,

Dempsey³

et al. 2014

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

328

382

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Photoactivatable fluorescent proteins (PAFPs) have been widely used for superresolution imaging based on the switching and localization of single molecules. Several properties of PAFPs strongly influence the quality of the superresolution images. These properties include (i) the number of photons emitted per switching cycle, which affects the localization precision of individual molecules; (ii) the ratio of the on-and off-switching rate constants, which limits the achievable localization density; (iii) the dimerization tendency, which could cause undesired aggregation of target proteins; and (iv) the signaling efficiency, which determines the fraction of target-PAFP fusion proteins that is detectable in a cell. Here, we evaluated these properties for 12 commonly used PAFPs fused to both bacterial target proteins, H-NS, HU, and Tar, and mammalian target proteins, Zyxin and Vimentin. Notably, none of the existing PAFPs provided optimal performance in all four criteria, particularly in the signaling efficiency and dimerization tendency. The PAFPs with low dimerization tendencies exhibited low signaling efficiencies, whereas mMaple showed the highest signaling efficiency but also a high dimerization tendency. To address this limitation, we engineered two new PAFPs based on mMaple, which we termed mMaple2 and mMaple3. These proteins exhibited substantially reduced or undetectable dimerization tendencies compared with mMaple but maintained the high signaling efficiency of mMaple. In the meantime, these proteins provided photon numbers and on-off switching rate ratios that are comparable to the best achieved values among PAFPs.P hotoactivated localization microscopy, stochastic optical reconstruction microscopy, and related imaging methods take advantage of photoswitching and imaging of single molecules to circumvent the diffraction limit of spatial resolution in light microscopy (1-3). In these methods, only a subset of the fluorescent labels in the sample is switched on at any given time such that the positions of individual fluorophores can be localized from their images with high precision. Iteration of this process allows numerous fluorescent labels to be localized and an image with sub-diffraction-limit resolution to be reconstructed from the fluorophore localizations. Fluorescent proteins that can be activated from dark to fluorescent or converted from one color to another are widely used for such imaging approaches (4, 5). Although photoactivatable fluorescent proteins (PAFPs) are generally dimmer than photoswitchable dyes (6, 7) and hence give lower image resolution, the ease and high specificity of labeling protein targets in living cells with fluorescent proteins makes PAFPs highly appealing probes for imaging the dynamics of cellular structures (4,8).For single-molecule-based superresolution imaging methods, several properties of PAFPs are particularly important for the image quality. Here, we focus on four such key properties. (i) The first property is the photon budget, defined as the average number of ph...

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“…H-NS exists primarily as a dimer at low concentrations but can multimerize into higher order complexes [4,5] that form bridges between adjacent DNA helices [6]. Optical tweezers have been used to demonstrate that H-NS dimers or multimers can simultaneously interact with separate DNA binding sites [7••].…”

Section: H-ns--a Nucleoid Proteinmentioning

confidence: 99%

“…Regulatory effects of supercoiling are linked to metabolic and environmental conditions. Thus, H-NS has been viewed as a nucleoid structuring protein with global effects on gene expression [3].H-NS exists primarily as a dimer at low concentrations but can multimerize into higher order complexes [4,5] that form bridges between adjacent DNA helices [6]. Optical tweezers have been used to demonstrate that H-NS dimers or multimers can simultaneously interact with separate DNA binding sites [7••].…”

mentioning

confidence: 99%

New insights into transcriptional regulation by H-NS

Fang

Rimsky

2008

Current Opinion in Microbiology

186

173

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H-NS, a nucleoid-associated DNA-binding protein of enteric bacteria, was discovered thirty-five years ago and subsequently found to exert widespread and highly pleiotropic effects on gene regulation. H-NS binds to high-affinity sites and spreads along adjacent AT-rich DNA to silence transcription. Preferential binding to sequences with higher AT-content than the resident genome allows H-NS to repress the expression of foreign DNA in a process known as "xenogeneic silencing." Counter-silencing by a variety of mechanisms facilitates the evolutionary acquisition of horizontally transferred genes and their integration into pre-existing regulatory networks. This review will highlight recent insights into the mechanism and biological importance of H-NS-DNA interactions. H-NS--A Nucleoid ProteinH-NS is an abundant DNA-binding protein implicated in the organization of the bacterial chromosome [1]. H-NS and other nucleoid-associated proteins can affect DNA topology at specific loci, thereby modulating gene transcription. Binding by these proteins is proposed to selectively direct supercoiling effects to promoters [2•]. Mutation of hns alters the responsiveness of transcription to changes in DNA superhelicity. Regulatory effects of supercoiling are linked to metabolic and environmental conditions. Thus, H-NS has been viewed as a nucleoid structuring protein with global effects on gene expression [3].H-NS exists primarily as a dimer at low concentrations but can multimerize into higher order complexes [4,5] that form bridges between adjacent DNA helices [6]. Optical tweezers have been used to demonstrate that H-NS dimers or multimers can simultaneously interact with separate DNA binding sites [7••]. H-NS-coated DNA is not self-interactive, suggesting that dimerization or oligomerization must precede DNA binding for bridging to occur. Force measurements suggest that transcription barriers created by H-NS binding are weak (∼7 pN) and readily overcome, so that H-NS oligomers pose only a relative barrier to translocating RNA polymerase. DNA bridging is a conserved feature of H-NS homologs found in most gram-negative bacteria [8] that appears to constrain large DNA loops [9••] and helps to account for the effects of H-NS on transcription. However, it must be noted that the relationship between bridging as a general effect and the silencing of specific promoters is not yet clear.H-NS is more appropriately viewed as a determinant of chromosomal architecture rather than as a general structural component. The analysis of nucleoids treated with urea suggests that *Corresponding Author : Tel 206-275-0966, Fax 206-616-1575, e-mail : fcfang@u.washington.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production proce...

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Oligomerization of the chromatin‐structuring protein H‐NS

Cited by 111 publications

References 32 publications

The Degree of Oligomerization of the H-NS Nucleoid Structuring Protein Is Related to Specific Binding to DNA

The Degree of Oligomerization of the H-NS Nucleoid Structuring Protein Is Related to Specific Binding to DNA

Characterization and development of photoactivatable fluorescent proteins for single-molecule–based superresolution imaging

New insights into transcriptional regulation by H-NS

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