Cognate Binding Sites Research Articles

Apolipoprotein L1 is transcriptionally regulated by SP1, IRF1 and IRF2 in hepatoma cells

Apolipoprotein L1 (APOL1) participates in lipid metabolism. Here, we investigate the mechanisms regulating APOL1 gene expression in hepatoma cells. We demonstrate that the -80-nt to +31-nt region of the APOL1 promoter, which contains one SP transcription factor binding GT box and an interferon regulatory factor (IRF) binding ISRE element, maintains the maximum activity. Mutation of the GT box and ISRE element dramatically reduces APOL1 promoter activity. EMSA and chromatin immunoprecipitation assay reveal that the transcription factors Sp1, IRF1 and IRF2 could interact with their cognate binding sites on the APOL1 promoter. Overexpression of Sp1, IRF1 and IRF2 increases promoter activity, leading to increased APOL1 mRNA and protein levels, while knockdown of Sp1, IRF1 and IRF2 has the opposite effects. These results demonstrate that the APOL1 gene could be regulated by Sp1, IRF1 and IRF2 in hepatoma cells.

RNA Binding Motif Protein RBM45 Regulates Expression of the 11-Kilodalton Protein of Parvovirus B19 through Binding to Novel Intron Splicing Enhancers.

During infection of human parvovirus B19 (B19V), one viral precursor mRNA (pre-mRNA) is transcribed by a single promoter and is alternatively spliced and alternatively polyadenylated. Here, we identified a novel cis-acting sequence (5'-GUA AAG CUA CGG GAC GGU-3'), intronic splicing enhancer 3 (ISE3), which lies 72 nucleotides upstream of the second splice acceptor (A2-2) site of the second intron that defines the exon of the mRNA encoding the 11-kDa viral nonstructural protein. RNA binding motif protein 45 (RBM45) specifically binds to ISE3 with high affinity (equilibrium dissociation constant [KD ] = 33 nM) mediated by its RNA recognition domain and 2-homo-oligomer assembly domain (RRM2-HOA). Knockdown of RBM45 expression or ectopic overexpression of RRM2-HOA in human erythroid progenitor cells (EPCs) expanded ex vivo significantly decreased the level of viral mRNA spliced at the A2-2 acceptor but not that of the mRNA spliced at A2-1 that encodes VP2. Moreover, silent mutations of ISE3 in an infectious DNA of B19V significantly reduced 11-kDa expression. Notably, RBM45 also specifically interacts in vitro with ISE2, which shares the octanucleotide (GGGACGGU) with ISE3. Taken together, our results suggest that RBM45, through binding to both ISE2 and ISE3, is an essential host factor for maturation of 11-kDa-encoding mRNA.IMPORTANCE Human parvovirus B19 (B19V) is a human pathogen that causes severe hematological disorders in immunocompromised individuals. B19V infection has a remarkable tropism with respect to human erythroid progenitor cells (EPCs) in human bone marrow and fetal liver. During B19V infection, only one viral precursor mRNA (pre-mRNA) is transcribed by a single promoter of the viral genome and is alternatively spliced and alternatively polyadenylated, a process which plays a key role in expression of viral proteins. Our studies revealed that a cellular RNA binding protein, RBM45, binds to two intron splicing enhancers and is essential for the maturation of the small nonstructural protein 11-kDa-encoding mRNA. The 11-kDa protein plays an important role not only in B19V infection-induced apoptosis but also in viral DNA replication. Thus, the identification of the RBM45 protein and its cognate binding site in B19V pre-mRNA provides a novel target for antiviral development to combat B19V infection-caused severe hematological disorders.

ChIPSummitDB: a ChIP-seq-based database of human transcription factor binding sites and the topological arrangements of the proteins bound to them

ChIP-seq reveals genomic regions where proteins, e.g. transcription factors (TFs) interact with DNA. A substantial fraction of these regions, however, do not contain the cognate binding site for the TF of interest. This phenomenon might be explained by protein–protein interactions and co-precipitation of interacting gene regulatory elements. We uniformly processed 3727 human ChIP-seq data sets and determined the cistrome of 292 TFs, as well as the distances between the TF binding motif centers and the ChIP-seq peak summits. ChIPSummitDB enables the analysis of ChIP-seq data using multiple approaches. The 292 cistromes and corresponding ChIP-seq peak sets can be browsed in GenomeView. Overlapping SNPs can be inspected in dbSNPView. Most importantly, the MotifView and PairShiftView pages show the average distance between motif centers and overlapping ChIP-seq peak summits and distance distributions thereof, respectively. In addition to providing a comprehensive human TF binding site collection, the ChIPSummitDB database and web interface allows for the examination of the topological arrangement of TF complexes genome-wide. ChIPSummitDB is freely accessible at http://summit.med.unideb.hu/summitdb/. The database will be regularly updated and extended with the newly available human and mouse ChIP-seq data sets.

Genome-wide investigation of WRKY transcription factors in sweet osmanthus and their potential regulation of aroma synthesis.

WRKY transcription factors, one of the largest transcription factor families, play important roles in regulating the synthesis of secondary metabolites. In sweet osmanthus (Osmanthus fragrans), the monoterpenes have been demonstrated as the most important volatile compounds, and the W-box, which is the cognate binding site of WRKY transcription factors, could be identified in most of the terpene-synthesis-related genes' promoters. However, the role of the WRKY family in terpene synthesis in sweet osmanthus has rarely been examined. In this study, 154 WRKY genes with conserved WRKY domain were identified and classified into three groups. The group II was further divided into five subgroups, and almost all members of IId contained a plant zinc cluster domain. Eight OfWRKYs (OfWRKY7/19/36/38/42/84/95/139) were screened from 20 OfWRKYs for their flower-specific expression patterns in different tissues. Simultaneously, the expression patterns of OfWRKYs and emission patterns of volatile compounds during the flowering process were determined and gas chromatography-mass spectrometry results showed that monoterpenes, such as linalool and ocimene, accounted for the highest proportion, contributing to the floral scent of sweet osmanthus in two cultivars. In addition, correlation analysis revealed the expression patterns of OfWRKYs (OfWRKY7/19/36/139) were each correlated with distinct monoterpenes (linalool, linalool derivatives, ocimene and ocimene derivatives). Subcellular localization analysis showed that p35S::GFP-OfWRKY7/38/95/139 were localized in the nucleus and OfWRKY139 had very strong transactivation activity. Collectively, the results indicated potential roles of OfWRKY139 and OfWRKYs with plant zinc cluster domain in regulating synthesis of aromatic compounds in sweet osmanthus, laying the foundation for use of OfWRKYs to improve the aroma of ornamental plants.

Guided nuclear exploration increases CTCF target search efficiency.

The enormous size of mammalian genomes means that for a DNA-binding protein, the number of non-specific, off-target sites vastly exceeds the number of specific, cognate sites. How mammalian DNA-binding proteins overcome this challenge to efficiently locate their target sites is not known. Here through live-cell single-molecule tracking, we show that CCCTC-binding factor, CTCF, is repeatedly trapped in small zones that likely correspond to CTCF clusters, in a manner that is largely dependent on an internal RNA-binding region (RBRi). We develop a new theoretical model, Anisotropic Diffusion through transient Trapping in Zones (ADTZ), to explain CTCF dynamics. Functionally, transient RBRi-mediated trapping increases the efficiency of CTCF target search by ~2.5 fold. Overall, our results suggest a “guided” mechanism where CTCF clusters concentrate diffusing CTCF proteins near cognate binding sites, thus increasing the local ON-rate. We suggest that local guiding may allow DNA-binding proteins to more efficiently locate their target sites.

Herpes simplex viral nucleoprotein creates a competitive transcriptional environment facilitating robust viral transcription and host shut off.

Herpes simplex virus-1 (HSV-1) replicates within the nucleus coopting the host's RNA Polymerase II (Pol II) machinery for production of viral mRNAs culminating in host transcriptional shut off. The mechanism behind this rapid reprogramming of the host transcriptional environment is largely unknown. We identified ICP4 as responsible for preferential recruitment of the Pol II machinery to the viral genome. ICP4 is a viral nucleoprotein which binds double-stranded DNA. We determined ICP4 discriminately binds the viral genome due to the absence of cellular nucleosomes and high density of cognate binding sites. We posit that ICP4's ability to recruit not just Pol II, but also more limiting essential components, such as TBP and Mediator, create a competitive transcriptional environment. These distinguishing characteristics ultimately result in a rapid and efficient reprogramming of the host's transcriptional machinery, which does not occur in the absence of ICP4.

An Assay for Quantifying Protein-RNA Binding in Bacteria.

In the initiation step of protein translation, the ribosome binds to the initiation region of the mRNA. Translation initiation can be blocked by binding of an RNA binding protein (RBP) to the initiation region of the mRNA, which interferes with ribosome binding. In the presented method, we utilize this blocking phenomenon to quantify the binding affinity of RBPs to their cognate and non-cognate binding sites. To do this, we insert a test binding site in the initiation region of a reporter mRNA and induce the expression of the test RBP. In the case of RBP-RNA binding, we observed a sigmoidal repression of the reporter expression as a function of RBP concentration. In the case of no-affinity or very low affinity between binding site and RBP, no significant repression was observed. The method is carried out in live bacterial cells, and does not require expensive or sophisticated machinery. It is useful for quantifying and comparing between the binding affinities of different RBPs that are functional in bacteria to a set of designed binding sites. This method may be inappropriate for binding sites with high structural complexity. This is due to the possibility of repression of ribosomal initiation by complex mRNA structure in the absence of RBP, which would result in lower basal reporter gene expression, and thus less-observable reporter repression upon RBP binding.

Ligity: A Non-Superpositional, Knowledge-Based Approach to Virtual Screening.

We present Ligity, a hybrid ligand-structure-based, non-superpositional method for virtual screening of large databases of small molecules. Ligity uses the relative spatial distribution of pharmacophoric interaction points (PIPs) derived from the conformations of small molecules. These are compared with the PIPs derived from key interaction features found in protein–ligand complexes and are used to prioritize likely binders. We investigated the effect of generating PIPs using the single lowest energy conformer versus an ensemble of conformers for each screened ligand, using different bin sizes for the distance between two features, utilizing triangular sets of pharmacophoric features (3-PIPs) versus chiral tetrahedral sets (4-PIPs), fusing data for targets with multiple protein–ligand complex structures, and applying different similarity measures. Ligity was benchmarked using the Directory of Useful Decoys-Enhanced (DUD-E). Optimal results were obtained using the tetrahedral PIPs derived from an ensemble of bound ligand conformers and a bin size of 1.5 Å, which are used as the default settings for Ligity. The high-throughput screening mode of Ligity, using only the lowest-energy conformer of each ligand, was used for benchmarking against the whole of the DUD-E, and a more resource-intensive, “information-rich” mode of Ligity, using a conformational ensemble of each ligand, were used for a representative subset of 10 targets. Against the full DUD-E database, mean area under the receiver operating characteristic curve (AUC) values ranged from 0.44 to 0.99, while for the representative subset they ranged from 0.61 to 0.86. Data fusion further improved Ligity’s performance, with mean AUC values ranging from 0.64 to 0.95. Ligity is very efficient compared to a protein–ligand docking method such as AutoDock Vina: if the time taken for the precalculation of Ligity descriptors is included in the comparason, then Ligity is about 20 times faster than docking. A direct comparison of the virtual screening steps shows Ligity to be over 5000 times faster. Ligity highly ranks the lowest-energy conformers of DUD-E actives, in a statistically significant manner, behavior that is not observed for DUD-E decoys. Thus, our results suggest that active compounds tend to bind in relatively low-energy conformations compared to decoys. This may be because actives—and thus their lowest-energy conformations—have been optimized for conformational complementarity with their cognate binding sites.

The Tat inhibitor didehydro-cortistatin A suppresses SIV replication and reactivation.

The HIV-1 transactivation protein (Tat) binds the HIV mRNA transactivation responsive element (TAR), regulating transcription and reactivation from latency. Drugs against Tat are unfortunately not clinically available. We reported that didehydro-cortistatin A (dCA) inhibits HIV-1 Tat activity. In human CD4+ T cells isolated from aviremic individuals and in the humanized mouse model of latency, combining dCA with antiretroviral therapy accelerates HIV-1 suppression and delays viral rebound upon treatment interruption. This drug class is amenable to block-and-lock functional cure approaches, aimed at a durable state of latency. Simian immunodeficiency virus (SIV) infection of rhesus macaques (RhMs) is the best-characterized model for AIDS research. Here, we demonstrate, using in vitro and cell-based assays, that dCA directly binds to SIV Tat's basic domain. dCA specifically inhibits SIV Tat binding to TAR, but not a Tat-Rev fusion protein, which activates transcription when Rev binds to its cognate RNA binding site replacing the apical region of TAR. Tat-TAR inhibition results in loss of RNA polymerase II recruitment to the SIV promoter. Importantly, dCA potently inhibits SIV reactivation from latently infected Hut78 cells and from primary CD4+ T cells explanted from SIVmac239-infected RhMs. In sum, dCA's remarkable breadth of activity encourages SIV-infected RhM use for dCA preclinical evaluation.-Mediouni, S., Kessing, C. F., Jablonski, J. A., Thenin-Houssier, S., Clementz, M., Kovach, M. D., Mousseau, G., de Vera, I.M.S., Li, C., Kojetin, D. J., Evans, D. T., Valente, S. T. The Tat inhibitor didehydro-cortistatin A suppresses SIV replication and reactivation.

An Exonic Switch Regulates Differential Accession of microRNAs to the Cd34 Transcript in Atherosclerosis Progression.

Background: CD34+ Endothelial Progenitor Cells (EPCs) play an important role in the recovery of injured endothelium and contribute to atherosclerosis (ATH) pathogenesis. Previously we described a potential atherogenic role for miR-125 that we aimed to confirm in this work. Methods: Microarray hybridization, TaqMan Low Density Array (TLDA) cards, qPCR, and immunohistochemistry (IHC) were used to analyze expression of the miRNAs, proteins and transcripts here studied. Results: Here we have demonstrated an increase of resident CD34-positive cells in the aortic tissue of human and mice during ATH progression, as well as the presence of clusters of CD34-positive cells in the intima and adventitia of human ATH aortas. We introduce miR-351, which share the seed sequence with miR-125, as a potential effector of CD34. We show a splicing event at an internal/cryptic splice site at exon 8 of the murine Cd34 gene (exonic-switch) that would regulate the differential accession of miRNAs (including miR-125) to the coding region or to the 3’UTR of Cd34. Conclusions: We introduce new potential mediators of ATH progression (CD34 cell-clusters, miR-351), and propose a new mechanism of miRNA action, linked to a cryptic splicing site in the target-host gene, that would regulate the differential accession of miRNAs to their cognate binding sites.

MiR-142-5p enhances cisplatin-induced apoptosis in ovarian cancer cells by targeting multiple anti-apoptotic genes

Chemotherapy is the preferred treatment for advanced ovarian cancer, but the 5-year survival rate remains low partly because of the development of drug resistance. Although it has been reported that X-linked inhibitor of apoptosis (XIAP) causes more severe chemoresistance in ovarian cancer cells and is highly expressed in chemoresistant ovarian cancer, the molecular mechanism underlying this dysregulation is unknown. The purpose of this study was to identify microRNAs (miRNAs) that bind to the 3′ untranslated region (3′UTR) of XIAP and have a role in chemoresistance in ovarian cancer. Using in silico analysis and literature review, a panel of miRNAs dysregulated in chemoresistant ovarian cancer was generated from hundreds of miRNAs that were predicted to target the XIAP 3′UTR. Using a dual luciferase reporter assay and cellular co-transfection of a miRNA expression vector and a luciferase reporter fused to the XIAP 3′UTR cognate miRNA binding site, we identified three miRNAs of which miR-142-5p had the greatest inhibitory effect. We found that overexpression of miR-142-5p suppressed XIAP expression by binding to its 3′UTR in OVCAR3 and SKOV3 cells. Using a chemosensitivity assay, we found that in OVCAR3, SKOV3, and primary epithelial ovarian cancer (EOC) cells, overexpression or inhibition of miR-142-5p increased or suppressed their sensitivities to cisplatin respectively. In contrast, introducing XIAP without a 3′UTR counteracted the effect of overexpressed miR-142-5p on cisplatin-induced apoptosis in OVCAR3 ovarian cancer cells. Furthermore, we found a negative correlation between miR-142-5p expression and XIAP protein levels in clinical samples from patients with EOC. Using clinical and miRNA expression data of more than 200 ovarian cancer patients treated with platinum-based chemotherapy from The Cancer Genome Atlas (TCGA) database, we found ovarian cancer patients with higher expression levels of miR-142-5p had longer median progression-free survival as compared to patients with lower miR-142-5p levels. We demonstrated that miR-142-5p also targeted four other anti-apoptotic genes, baculoviral IAP repeat-containing 3 (BIRC3), B-cell lymphoma-2 (BCL2), BCL2 like 2 (BCL2L2), and myeloid cell leukemia sequence 1 (MCL1) specifically. Transcriptome sequencing shed light on the essential apoptosis-related pathway in which miR-142-5p may be involved. To conclude, our findings illustrate that miR-142-5p sensitizes ovarian cancer cells to cisplatin-induced apoptosis by targeting multiple anti-apoptotic genes including XIAP, and may also suggest the therapeutic potential of miR-142-5p in ovarian cancer treatment.

Protein-protein binding supersites.

The lack of a deep understanding of how proteins interact remains an important roadblock in advancing efforts to identify binding partners and uncover the corresponding regulatory mechanisms of the functions they mediate. Understanding protein-protein interactions is also essential for designing specific chemical modifications to develop new reagents and therapeutics. We explored the hypothesis of whether protein interaction sites serve as generic biding sites for non-cognate protein ligands, just as it has been observed for small-molecule-binding sites in the past. Using extensive computational docking experiments on a test set of 241 protein complexes, we found that indeed there is a strong preference for non-cognate ligands to bind to the cognate binding site of a receptor. This observation appears to be robust to variations in docking programs, types of non-cognate protein probes, sizes of binding patches, relative sizes of binding patches and full-length proteins, and the exploration of obligate and non-obligate complexes. The accuracy of the docking scoring function appears to play a role in defining the correct site. The frequency of interaction of unrelated probes recognizing the binding interface was utilized in a simple prediction algorithm that showed accuracy competitive with other state of the art methods.

The key protein of endosomal mRNP transport Rrm4 binds translational landmark sites of cargo mRNAs.

RNA-binding proteins (RBPs) determine spatiotemporal gene expression by mediating active transport and local translation of cargo mRNAs. Here, we cast a transcriptome-wide view on the transported mRNAs and cognate RBP binding sites during endosomal messenger ribonucleoprotein (mRNP) transport in Ustilago maydis Using individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP), we compare the key transport RBP Rrm4 and the newly identified endosomal mRNP component Grp1 that is crucial to coordinate hyphal growth. Both RBPs bind predominantly in the 3' untranslated region of thousands of shared cargo mRNAs, often in close proximity. Intriguingly, Rrm4 precisely binds at stop codons, which constitute landmark sites of translation, suggesting an intimate connection of mRNA transport and translation. Towards uncovering the code of recognition, we identify UAUG as specific binding motif of Rrm4 that is bound by its third RRM domain. Altogether, we provide first insights into the positional organisation of co-localising RBPs on individual cargo mRNAs.

An in Vivo Binding Assay for RNA-Binding Proteins Based on Repression of a Reporter Gene

We study translation repression in bacteria by engineering a regulatory circuit that functions as a binding assay for RNA binding proteins (RBP) in vivo. We do so by inducing expression of a fluorescent protein-RBP chimera, together with encoding its binding site at various positions within the ribosomal initiation region (+11-13 nt from the AUG) of a reporter module. We show that when bound by their cognate RBPs, the phage coat proteins for PP7 (PCP) and Qβ (QCP), strong repression is observed for all hairpin positions within the initiation region. Yet, a sharp transition to no-effect is observed when positioned in the elongation region, at a single-nucleotide resolution. Employing in vivo Selective 2'-hydroxyl acylation analyzed by primer extension followed by sequencing (SHAPE-seq) for a representative construct, established that in the translationally active state the mRNA molecule is nonstructured, while in the repressed state a structured signature was detected. We then utilize this regulatory phenomena to quantify the binding affinity of the coat proteins of phages MS2, PP7, GA, and Qβ to 14 cognate and noncognate binding sites in vivo. Using our circuit, we demonstrate qualitative differences between in vitro to in vivo binding characteristics for various variants when comparing to past studies. Furthermore, by introducing a simple mutation to the loop region for the Qβ-wt site, MCP binding is abolished, creating the first high-affinity QCP site that is completely orthogonal to MCP. Consequently, we demonstrate that our hybrid transcriptional-post-transcriptional circuit can be utilized as a binding assay to quantify RNA-RBP interactions in vivo.

Crystal structure of TraN, a regulator of conjugative DNA transfer, in complex with its cognate DNA binding site

Crystal structure of TraN, a regulator of conjugative DNA transfer, in complex with its cognate DNA binding site

A zinc-finger fusion protein refines Gal4-defined neural circuits

The analysis of behavior requires that the underlying neuronal circuits are identified and genetically isolated. In several major model species—most notably Drosophila—neurogeneticists identify and isolate neural circuits with a binary heterologous expression-control system: Gal4–UASG. One limitation of Gal4–UASG is that expression patterns are often too broad to map circuits precisely. To help refine the range of Gal4 lines, we developed an intersectional genetic AND operator. Interoperable with Gal4, the new system’s key component is a fusion protein in which the DNA-binding domain of Gal4 has been replaced with a zinc finger domain with a different DNA-binding specificity. In combination with its cognate binding site (UASZ) the zinc-finger-replaced Gal4 (‘Zal1’) was functional as a standalone transcription factor. Zal1 transgenes also refined Gal4 expression ranges when combined with UASGZ, a hybrid upstream activation sequence. In this way, combining Gal4 and Zal1 drivers captured restricted cell sets compared with single drivers and improved genetic fidelity. This intersectional genetic AND operation presumably derives from the action of a heterodimeric transcription factor: Gal4-Zal1. Configurations of Zal1–UASZ and Zal1-Gal4-UASGZ are versatile tools for defining, refining, and manipulating targeted neural expression patterns with precision.

A functional in vitro assay for screening inhibitors of STAT5B phosphorylation

Inhibition of STAT phosphorylation is recognized as a viable therapeutic strategy for disrupting tumorigenesis. Constitutive STAT phosphorylation is found with high frequency in a number of primary tumor types, while non-cancer cells exhibit low basal activity, providing an exploitable therapeutic window. STAT activation involves phosphorylation of the SH2 domain by a number of tyrosine kinases followed by STAT dimerization and translocation to the nucleus. By blocking the cognate binding site, STAT SH2-domain inhibitors can impede kinase-mediated de novo STAT phosphorylation. Assessing for inhibitors of STAT phosphorylation has previously been conducted exclusively in cellulo using Western blot analysis. However, while providing useful in cellulo efficacy, it is not possible to conclude that inhibition is due to a direct blockade of STAT protein. Here we developed a functional assay that directly reports the blockade of phosphorylation as a result of inhibitor interaction with STAT proteins. We have optimized reaction conditions for the functional assay and validated the assay against known STAT5B ligands, including peptides and small molecule inhibitors. As part of the study, we have also identified several sites of STAT5B phosphorylation by Abl kinase. This assay will serve to delineate the functional mechanism of STAT binders in vitro and deconvolute the mechanism of phospho-STAT inhibition observed in Western blot analysis.

Characterization of Small Molecules Inhibiting the Pro-Angiogenic Activity of the Zinc Finger Transcription Factor Vezf1.

Discovery of inhibitors for endothelial-related transcription factors can contribute to the development of anti-angiogenic therapies that treat various diseases, including cancer. The role of transcription factor Vezf1 in vascular development and regulation of angiogenesis has been defined by several earlier studies. Through construction of a computational model for Vezf1, work here has identified a novel small molecule drug capable of inhibiting Vezf1 from binding to its cognate DNA binding site. Using structure-based design and virtual screening of the NCI Diversity Compound Library, 12 shortlisted compounds were tested for their ability to interfere with the binding of Vezf1 to DNA using electrophoretic gel mobility shift assays. We identified one compound, T4, which has an IC50 of 20 μM. Using murine endothelial cells, MSS31, we tested the effect of T4 on endothelial cell viability and angiogenesis by using tube formation assay. Our data show that addition of T4 in cell culture medium does not affect cell viability at concentrations lower or equal to its IC 50 but strongly inhibits the network formation by MSS31 in the tube formation assays. Given its potential efficacy, this inhibitor has significant therapeutic potential in several human diseases.

Structure-guided approach to site-specific fluorophore labeling of the lac repressor LacI.

The lactose operon repressor protein LacI has long served as a paradigm of the bacterial transcription factors. However, the mechanisms whereby LacI rapidly locates its cognate binding site on the bacterial chromosome are still elusive. Single-molecule fluorescence imaging approaches are well suited for the study of these mechanisms but rely on a functionally compatible fluorescence labeling of LacI. Particularly attractive for protein fluorescence labeling are synthetic fluorophores due to their small size and favorable photophysical characteristics. Synthetic fluorophores are often conjugated to natively occurring cysteine residues using maleimide chemistry. For a site-specific and functionally compatible labeling with maleimide fluorophores, the target protein often needs to be redesigned to remove unwanted native cysteines and to introduce cysteines at locations better suited for fluorophore attachment. Biochemical screens can then be employed to probe for the functional activity of the redesigned protein both before and after dye labeling. Here, we report a mutagenesis-based redesign of LacI to enable a functionally compatible labeling with maleimide fluorophores. To provide an easily accessible labeling site in LacI, we introduced a single cysteine residue at position 28 in the DNA-binding headpiece of LacI and replaced two native cysteines with alanines where derivatization with bulky substituents is known to compromise the protein’s activity. We find that the redesigned LacI retains a robust activity in vitro and in vivo, provided that the third native cysteine at position 281 is retained in LacI. In a total internal reflection microscopy assay, we observed individual Cy3-labeled LacI molecules bound to immobilized DNA harboring the cognate O1 operator sequence, indicating that the dye-labeled LacI is functionally active. We have thus been able to generate a functional fluorescently labeled LacI that can be used to unravel mechanistic details of LacI target search at the single molecule level.

Modulation of H-NS transcriptional silencing by magnesium.

The bacterial histone-like protein H-NS silences AT-rich DNA, binding DNA as ‘stiffened’ filaments or ‘bridged’ intrastrand loops. The switch between these modes has been suggested to depend on the concentration of divalent cations, in particular Mg2+, with elevated Mg2+ concentrations associated with a transition to bridging. Here we demonstrate that the observed binding mode is a function of the local concentration of H-NS and its cognate binding sites, as well as the affinity of the interactions between them. Mg2+ does not control a binary switch between these two modes but rather modulates the affinity of this interaction, inhibiting the DNA-binding and silencing activity of H-NS in a continuous linear fashion. The direct relationship between conditions that favor stiffening and transcriptional silencing activity suggests that although both modes can occur in the cell, stiffening is the predominant mode of binding at silenced genes.