Cognate Binding Sites Research Articles

RUNX1 regulates promoter activity in the absence of cognate DNA binding motifs.

Runt-related transcription factor 1 (RUNX1) plays an important role in normal haematopoietic cell development and function, and its function is frequently disrupted in leukaemia. RUNX1 is widely recognised as a sequence-specific DNA binding factor that recognises the motif 5'-TG(T/C)GGT-3' in promoter and enhancer regions of its target genes. Moreover, RUNX1 fusion proteins, such as RUNX1-ETO formed by the t(8;21) translocation, retain the ability to recognise and bind to this sequence to elicit atypical gene regulatory effects on bona fide RUNX1 targets. However, our analysis of publicly available RUNX1 chromatin immunoprecipitation sequencing (ChIP-Seq) data has provided evidence challenging this dogma, revealing that this motif-specific model of RUNX1 recruitment and function is incomplete. Our analyses revealed that the majority of RUNX1 genomic localisation occurs outside of promoters, that 20% of RUNX1 binding sites lack consensus RUNX motifs, and that binding in the absence of a cognate binding site is more common in promoter regions compared to distal sites. Reporter assays demonstrate that RUNX1 can drive promoter activity in the absence of a recognised DNA binding motif, in contrast to RUNX1-ETO. RUNX1-ETO supresses activity when it is recruited to promoters containing a sequence specific motif, while interestingly, it binds but does not repress promoters devoid of a RUNX1 recognition site. These data suggest that RUNX1 regulation of target genes occurs through multiple mechanisms depending on genomic location, the type of regulatory element and mode of recruitment.

The Enrichment of miRNA-Targeted mRNAs in Translationally Less Active over More Active Polysomes.

miRNAs moderately inhibit the translation and enhance the degradation of their target mRNAs via cognate binding sites located predominantly in the 3'-untranslated regions (UTR). Paradoxically, miRNA targets are also polysome-associated. We studied the polysome association by the comparative translationally less-active light- and more-active heavy-polysome profiling of a wild type (WT) human cell line and its isogenic mutant (MT) with a disrupted DICER1 gene and, thus, mature miRNA production. As expected, the open reading frame (ORF) length is a major determinant of light- to heavy-polysome mRNA abundance ratios, but is rendered less powerful in WT than in MT cells by miRNA-regulatory activities. We also observed that miRNAs tend to target mRNAs with longer ORFs, and that adjusting the mRNA abundance ratio with the ORF length improves its correlation with the 3'-UTR miRNA-binding-site count. In WT cells, miRNA-targeted mRNAs exhibit higher abundance in light relative to heavy polysomes, i.e., light-polysome enrichment. In MT cells, the DICER1 disruption not only significantly abrogated the light-polysome enrichment, but also narrowed the mRNA abundance ratio value range. Additionally, the abrogation of the enrichment due to the DICER1 gene disruption, i.e., the decreases of the ORF-length-adjusted mRNA abundance ratio from WT to MT cells, exhibits a nearly perfect linear correlation with the 3'-UTR binding-site count. Transcription factors and protein kinases are the top two most enriched mRNA groups. Taken together, the results provide evidence for the light-polysome enrichment of miRNA-targeted mRNAs to reconcile polysome association and moderate translation inhibition, and that ORF length is an important, though currently under-appreciated, transcriptome regulation parameter.

Lambda CI Binding to Related Phage Operator Sequences Validates Alignment Algorithm and Highlights the Importance of Overlooked Bonds.

Bacteriophage λ's CI repressor protein controls a genetic switch between the virus's lysogenic and lytic lifecycles, in part, by selectively binding to six different DNA sequences within the phage genome-collectively referred to as operator sites. However, the minimal level of information needed for CI to recognize and specifically bind these six unique-but-related sequences is unclear. In a previous study, we introduced an algorithm that extracts the minimal direct readout information needed for λ-CI to recognize and bind its six binding sites. We further revealed direct readout information shared among three evolutionarily related lambdoid phages: λ-phage, Enterobacteria phage VT2-Sakai, and Stx2 converting phage I, suggesting that the λ-CI protein could bind to the operator sites of these other phages. In this study, we show that λ-CI can indeed bind the other two phages' cognate binding sites as predicted using our algorithm, validating the hypotheses from that paper. We go on to demonstrate the importance of specific hydrogen bond donors and acceptors that are maintained despite changes to the nucleobase itself, and another that has an important role in recognition and binding. This in vitro validation of our algorithm supports its use as a tool to predict alternative binding sites for DNA-binding proteins.

Mechanism of δ Mediated Transcription Activation in Bacillus subtilis: Interaction with α CTD of RNA Polymerase Stabilizes δ and Successively Facilitates the Open Complex Formation

The α CTD (C-terminal domain of the α subunit) of RNA polymerase (RNAP) is a target for transcriptional regulators. In the transcription activation at Class I, Class II, and Class III promoters of bacteria, the transcriptional regulator, binds to DNA at different sites and interacts with the α CTD to stabilize the RNAP at the promoter or it binds to the α CTD to form a prerecruitment complex that searches for its cognate binding site. This ‘simple recruitment mechanism’ of the transcriptional machinery at the promoter is responsible for the activation of transcription. Strikingly, in B. subtilis the binding of RNAP at the promoter stabilizes the transcriptional regulator, δ at the −41 site of the promoter DNA through an interaction with its α CTD and successively facilitates the open complex formation. Two residues R293 and K294 of α CTD (equivalent to K297 and K298 of E. coli) are involved in the interactions with δ and essential for the activation of transcription. R293 is responsible for the stabilization of δ, while K294 is responsible for facilitating the open complex formation. Based on our data we propose a new model of transcription activation by δ of B. subtilis that is similar to (its binding location and interaction with α CTD), but distinct from (the recruitment of transcription factor by RNAP at the DNA, and enhancement of the open complex formation) the model Class II promoters in bacteria.

Modulation of protein-DNA binding reveals mechanisms of spatiotemporal gene control in early Drosophila embryos.

It is well known that enhancers regulate the spatiotemporal expression of their target genes by recruiting transcription factors (TFs) to the cognate binding sites in the region. However, the role of multiple binding sites for the same TFs and their specific spatial arrangement in determining the overall competency of the enhancer has yet to be fully understood. In this study, we utilized the MS2-MCP live imaging technique to quantitatively analyze the regulatory logic of the snail distal enhancer in early Drosophila embryos. Through systematic modulation of Dorsal and Twist binding motifs in this enhancer, we found that a mutation in any one of these binding sites causes a drastic reduction in transcriptional amplitude, resulting in a reduction in mRNA production of the target gene. We provide evidence of synergy, such that multiple binding sites with moderate affinities cooperatively recruit more TFs to drive stronger transcriptional activity than a single site. Moreover, a Hidden Markov-based stochastic model of transcription reveals that embryos with mutated binding sites have a higher probability of returning to the inactive promoter state. We propose that TF-DNA binding regulates spatial and temporal gene expression and drives robust pattern formation by modulating transcriptional kinetics and tuning bursting rates.

Momordica anti-HIV protein MAP30 abrogates the Epstein-Barr virus nuclear antigen 1 dependent functions in host cells

Originally extracted from Momordica charantia seeds, the antiviral and anti-tumor activities of Momordica anti-HIV protein MAP30 have become well known. Although MAP30 has been reported to possess antiviral activity against several human viruses, the current understanding of the MAP30-mediated antiviral response is mainly derived from the previous research work on anti-HIV herbal medicines; the mechanistic insight of its effects on other viruses remains largely unknown. In this study, we showed that both ectopically expressed and purified recombinant MAP30 (rMAP30) impeded Epstein-Barr virus Nuclear Antigen 1 (EBNA1)-mediated transcription from the viral latent replication origin. Mechanistically, in vivo and in vitro studies revealed that MAP30 caused EBNA1 to dissociate from the cognate binding sites, which disrupted downstream EBNA1-dependent viral epigenome accumulation and cell maintenance of Epstein-Barr virus (EBV)-associated neoplastic cells. Finally, mutational analysis indicated that the N-terminal ricin A homologous domain shared by ricin-like proteins was implicated in the anti-EBV response. Our study provides evidence to support that MAP30 has a unique property to combat EBV latent infection, suggesting a potential to develop this herbal protein to be an alternative medicine for EBV associated diseases.

Sequence-Dependent Interaction of the Human Papillomavirus E2 Protein with the DNA Elements on Its DNA Replication Origin.

The human papillomavirus (HPV) E2 protein is essential for regulating the initiation of viral DNA replication as well as the regulation of transcription of certain HPV-encoded genes. Its ability to recognize and bind to its four recognition sequences in the viral origin is a key step in the initiation of HPV DNA replication. Thus, understanding the mechanism of DNA binding by E2 protein and the unique roles played by individual DNA sequence elements of the replication origin is essential. We have purified the recombinant full-length HPV type 11 E2 protein. Quantitative DNA binding analysis indicated E2 protein bound all four DNA binding sites with reasonably high affinities but with distinct preferences. It bound its cognate binding sites 1, 2, and 4 with higher affinities, but bound binding site 3 with lower affinity. Analysis of binding to these sites unraveled multiple sequence elements that appeared to influence E2 binding affinity and target discrimination, including the sequence of spacer region, flanking sequences, and proximity of E2 binding sites. Thermodynamic analysis indicated hydrophobic interaction in the protein-DNA complex formation. Our studies indicate a large multi-protein complex formation on the HPV-origin DNA, likely due to reasonably high binding affinities as well as intrinsic oligomerization propensity of E2 dimers.

The HP1α protein is mandatory to repress the circadian clock and its output genes during the 12 h period of transcriptional repression

The transcriptional repressions driven by the circadian core clock repressors RevErbα, E4BP4, and CRY1/PER1 involve feedback loops which are mandatory for generating the circadian rhythms. These repressors are known to bind to cognate DNA binding sites, but how their circadian bindings trigger the cascade of events leading to these repressions remain to be elucidated. Through molecular and genetic analyses, we now demonstrate that the chromatin protein HP1α plays a key role in these transcriptional repressions of both the circadian clock (CC) genes and their cognate output genes (CCGs). We show that these CC repressors recruit the HP1α protein downstream from a repressive cascade, and that this recruitment is mandatory for the maintenance of both the CC integrity and the expression of the circadian genes. We further show that the presence of HP1α is critical for both the repressor-induced chromatin compaction and the generation of "transcriptionally repressed biomolecular hydrophobic condensates" and demonstrates that HP1α is mandatory within the CC output genes for both the recruitment of DNA methylating enzymes on the intronic deoxyCpG islands and their subsequent methylation.

Demixing of distinct ribonucleoprotein condensates is under dynamical control.

Whi3 is an RNA binding protein from Ashbya gossypii that phase separates to form RNA-protein condensates. Observations in cells show that certain RNA molecules co-localize with Whi3 and other RNA molecules into a single condensate, while some others form apparently demixed condensates. Is a there a thermodynamic basis for different ternary combinations of Whi3 and RNA molecules to form demixed condensates? Simulations show that RNA molecules must have strong repulsive interactions for one another, or strong homotypic interactions if demixing is to be thermodynamically feasible. We tested this using in vitro reconstitutions to quantify the phase behaviors of binary and ternary mixtures. A novel analysis of the measured phase boundaries show that heterotypic protein-RNA interactions are the main drivers of phase separation. This was further tested by deletion of cognate binding sites on RNA molecules, showing that there are several ways for Whi3 to associate with RNA molecules. Investigations of phase behaviors internary mixtures show that demixed condensates are dynamically arrested phases. Therefore, in vitro studies suggest that demixing is likely to be under dynamical rather than thermodynamic control. To test for this in live cells, we generated a mutant where differentially expressed RNAs are constitutively expressed under a single promoter. We find that RNA molecules that demix in a wildtype background formed colocalized condensates when they are expressed concomitantly. Our studies show that the non-equilibrium aspects of phase separation, specifically the ability to form dynamically arrested phases, can give rise to demixed condensates that achieve long-livedspatial segregation of RNA molecules within distinct condensates.

Annotation of biologically relevant ligands in UniProtKB using ChEBI.

To provide high quality, computationally tractable annotation of binding sites for biologically relevant (cognate) ligands in UniProtKB using the chemical ontology ChEBI (Chemical Entities of Biological Interest), to better support efforts to study and predict functionally relevant interactions between protein sequences and structures and small molecule ligands. We structured the data model for cognate ligand binding site annotations in UniProtKB and performed a complete reannotation of all cognate ligand binding sites using stable unique identifiers from ChEBI, which we now use as the reference vocabulary for all such annotations. We developed improved search and query facilities for cognate ligands in the UniProt website, REST API and SPARQL endpoint that leverage the chemical structure data, nomenclature and classification that ChEBI provides. Binding site annotations for cognate ligands described using ChEBI are available for UniProtKB protein sequence records in several formats (text, XML and RDF) and are freely available to query and download through the UniProt website (www.uniprot.org), REST API (www.uniprot.org/help/api), SPARQL endpoint (sparql.uniprot.org/) and FTP site (https://ftp.uniprot.org/pub/databases/uniprot/). Supplementary data are available at Bioinformatics online.

Sulfane Sulfur Is an Intrinsic Signal for the Organic Peroxide Sensor OhrR of Pseudomonas aeruginosa.

Sulfane sulfur, including organic persulfide and polysulfide, is a normal cellular component, and its level varies during growth. It is emerging as a signaling molecule in bacteria, regulating the gene regulator MarR in Escherichia coli, MexR in Pseudomonas aeruginosa, and MgrA of Staphylococcus aureus. They are MarR-family regulators and are often repressors for multiple antibiotic resistance genes. Here, we report that another MarR-type regulator OhrR that represses the expression of itself and a thiol peroxidase gene ohr in P. aeruginosa PAO1 also responded to sulfane sulfur. PaOhrR formed disulfide bonds between three Cys residues within a dimer after polysulfide treatment. The modification reduced its affinity to its cognate DNA binding site. An Escherichia coli reporter system, in which mKate was under the repression of OhrR, showed that PaOhrR derepressed its controlled gene when polysulfide was added, whereas the mutant PaOhrR with two Cys residues changed to Ser residues did not respond to polysulfide. The expression of the PaOhrR-repressed mKate was significantly increased when the cells enter the late log phase when cellular sulfane sulfur reached a maximum, but the mKate expression under the control of the PaOhrR-C9SC19S double mutant was not increased. Furthermore, the expression levels of ohrR and ohr in P. aeruginosa PAO1 were significantly increased when cellular sulfane sulfur was high. Thus, PaOhrR senses both exogenous and intrinsic sulfane sulfur to derepress its controlled genes. The finding also suggests that sulfane sulfur may be a common inducer of the MarR-type regulators, which may confer the bacteria to resist certain stresses without being exposed to the stresses.

Flexibility of flanking DNA is a key determinant of transcription factor affinity for the core motif

Selective gene regulation is mediated by recognition of specific DNA sequences by transcription factors (TFs). The extremely challenging task of searching out specific cognate DNA binding sites among several million putative sites within the eukaryotic genome is achieved by complex molecular recognition mechanisms. Elements of this recognition code include the core binding sequence, the flanking sequence context, and the shape and conformational flexibility of the composite binding site. To unravel the extent to which DNA flexibility modulates TF binding, in this study, we employed experimentally guided molecular dynamics simulations of ternary complex of closely related Hox heterodimers Exd-Ubx and Exd-Scr with DNA. Results demonstrate that flexibility signatures embedded in the flanking sequences impact TF binding at the cognate binding site. A DNA sequence has intrinsic shape and flexibility features. While shape features are localized, our analyses reveal that flexibility features of the flanking sequences percolate several basepairs and allosterically modulate TF binding at the core. We also show that lack of flexibility in the motif context can render the cognate site resistant to protein-induced shape changes and subsequently lower TF binding affinity. Overall, this study suggests that flexibility-guided DNA shape, and not merely the static shape, is a key unexplored component of the complex DNA-TF recognition code.

Protein‐DNA Interactomes of NKX2‐5 and TBX5 Mutants Identified in Congenital Heart Defects

Congenital Heart Defects (CHDs) are the most common birth defect from children to adults in the United States. CHDs are characterized by malformations in the heart’s chambers, walls, and great vessels, sometimes leading to embryonic death. Cardiac Transcription Factors (cardiac TFs) such as NKX2‐5 and TBX5 play a critical role in developmental stages and physiological processes of the heart by regulating gene expression through DNA‐protein interactions. Genetic analyses have identified various non‐synonymous mutations within the homeodomain (HD) DNA‐binding domain (DBD) of NKX2‐5 and the (Tbox) DBD of TBX5 in associated with CHD. We hypothesize that non‐synonymous mutations in cardiac TFs DBD will alter their DNA‐binding properties ultimately affecting transcriptional pathways necessary for proper heart development. To address this hypothesis, we studied how the cardiac TFs DNA‐protein interactomes are affected by non‐synonymous mutations found in CHD patients. We expressed and purified the DBD of NKX2‐5 (HD), TBX5 (Tbox), also NKX2‐5 and TBX5 non‐synonymous mutations (A148E, E154G, R161P, T178M, Q181H, R190C, Y191C), and (I54T, M74V, G80R, I101F, R113K, R237Q, R237W) respectively. Functional analysis using Electrophoretic Mobility Shift Assay (EMSA) confirms HD and Tbox binding to cognate DNA binding sites. DNA‐binding activity as assessed by EMSA, shows retention of DNA activity in the case of NKX2‐5 and TBX5 mutants A148E, R161P, Y191C and I54T, R113K respectively. Mutants T178M, Q181H, R190C, M74V, G80R, I101F, R237Q, R237W showed complete loss in DNA binding activity towards their specific cognate sites. We measured the comprehensive protein‐DNA interactomes of NKX2‐5 and TBX5 non‐synonymous mutants using High‐Throughput Systematic Evolution of Ligands and Exponential Enrichment (SELEX‐seq) to identify changes in DNA targets between wild‐type and mutant TFs. Mutants A148E, R161P and Y191C bind to similar binding sites as NKX2‐5 (HD). Interestingly, mutant Y191C showed an altered DNA binding site significantly different from NKX2‐5 (HD). Mutants I54T, R113K bind to similar binding sites as TBX5 (Tbox). Mutants E154G, T178M, Q181H, R190C, M74V, G80R, I101F, R237Q and R237W showed no significant enrichment for specific DNA binding sites. Integrative analysis with available NKX2‐5 and TBX5 genomic binding sites data (ChIP‐seq), human and mouse cardiac enhancers and promoters catalogued in the literature will facilitate the identification of putative genes mutant cardiac TFs are regulating and how different are from their wild‐type TFs.

A detailed investigation to study the pattern of the interplay of Cyclic AMP Receptor Protein (CRP) of E. coli with its different classes of promoters

The activity of most of the promoters in Escherichia coli, involved in the metabolism of sugars other than glucose, is controlled by a CRP (cAMP receptor protein) or CAP (catabolite activator protein). CRP-dependent promoters are differentiated into various classes (Class I, Class II, and Class III) based on its cognate binding site’s position on DNA. The promoters regulated by CAP are differentially regulated by this transcriptional factor and it is also imperative to mention that these promoters vary greatly in respect to the binding site of CAP to its cognate binding site, it has also been reported that either it overlaps with the binding site of RNA polymerase or it present upstream to it. In Class I CAP-dependent promoters, a particular CAP molecule makes protein-protein interaction for the start of transcription. In Class II CAP-dependent promoters, a particular CAP molecule makes multiple interactions for the start of transcription. At last, in Class III-CAP dependent promoters, more than one CAP molecule is involved and activation of transcription is done synergistically. It has also been documented that CAP shows a kind of biphasic behavior in some promoters. So, the main focus of this work is to find out whether this biphasic behavior is true for other E. coli promoters as well. Experiments have been performed to know more about this biphasic nature and the various patterns of interactions of catabolite activator protein (CAP) of E. coli with its different classes of promoters.

RBPmap: A Tool for Mapping and Predicting the Binding Sites of RNA-Binding Proteins Considering the Motif Environment.

RNA-binding proteins (RBPs) play a key role in post-transcriptional regulation via binding to coding and non-coding RNAs. Recent development in experimental technologies, aimed to identify the targets of RBPs, has significantly broadened our knowledge on protein-RNA interactions. However, for many RBPs in many organisms and cell types, experimental RNA-binding data is not available. In this chapter we describe a computational approach, named RBPmap, available as a web service via http://rbpmap.technion.ac.il/ and as a stand-alone version for download. RBPmap was designed for mapping and predicting the binding sites of any RBP within a nucleic acid sequence, given the availability of an experimentally defined binding motif of the RBP. The algorithm searches for a sub-sequence that significantly matches the RBP motif, considering the clustering propensity of other weak matches within the motif environment. Here, we present different applications of RBPmap for discovering the involvement of RBPs and their targets in a variety of cellular processes, in health and disease states. Finally, we demonstrate the performance of RBPmap in predicting the binding targets of RBPs in large-scale RNA-binding data, reinforcing the strength of the tool in distinguishing cognate binding sites from weak motifs.

Liquid condensation of reprogramming factor KLF4 with DNA provides a mechanism for chromatin organization

Expression of a few master transcription factors can reprogram the epigenetic landscape and three-dimensional chromatin topology of differentiated cells and achieve pluripotency. During reprogramming, thousands of long-range chromatin contacts are altered, and changes in promoter association with enhancers dramatically influence transcription. Molecular participants at these sites have been identified, but how this re-organization might be orchestrated is not known. Biomolecular condensation is implicated in subcellular organization, including the recruitment of RNA polymerase in transcriptional activation. Here, we show that reprogramming factor KLF4 undergoes biomolecular condensation even in the absence of its intrinsically disordered region. Liquid–liquid condensation of the isolated KLF4 DNA binding domain with a DNA fragment from the NANOG proximal promoter is enhanced by CpG methylation of a KLF4 cognate binding site. We propose KLF4-mediated condensation as one mechanism for selectively organizing and re-organizing the genome based on the local sequence and epigenetic state.

Artificial Transcription Factors for Tuneable Gene Expression in Pichia pastoris.

The non-conventional yeast Pichia pastoris (syn. Komagataella phaffii) has become a powerful eukaryotic expression platform for biopharmaceutical and biotechnological applications on both laboratory and industrial scales. Despite the fundamental role that artificial transcription factors (ATFs) play in the orthogonal control of gene expression in synthetic biology, a limited number of ATFs are available for P. pastoris. To establish orthogonal regulators for use in P. pastoris, we characterized ATFs derived from Arabidopsis TFs. The plant-derived ATFs contain the binding domain of TFs from the plant Arabidopsis thaliana, in combination with the activation domains of yeast GAL4 and plant EDLL and a synthetic promoter harboring the cognate cis-regulatory motifs. Chromosomally integrated ATFs and their binding sites (ATF/BSs) resulted in a wide spectrum of inducible transcriptional outputs in P. pastoris, ranging from as low as 1- to as high as ∼63-fold induction with only small growth defects. We demonstrated the application of ATF/BSs by generating P. pastoris cells that produce β-carotene. Notably, the productivity of β-carotene in P. pastoris was ∼4.8-fold higher than that in S. cerevisiae, reaching ∼59% of the β-carotene productivity obtained in a S. cerevisiae strain optimized for the production of the β–carotene precursor, farnesyl diphosphate, by rewiring the endogenous metabolic pathways using plant-derived ATF/BSs. Our data suggest that plant-derived regulators have a high degree of transferability from S. cerevisiae to P. pastoris. The plant-derived ATFs, together with their cognate binding sites, powerfully increase the repertoire of transcriptional regulatory modules for the tuning of protein expression levels required in metabolic engineering or synthetic biology in P. pastoris.

Machine learning predicts nucleosome binding modes of transcription factors

BackgroundMost transcription factors (TFs) compete with nucleosomes to gain access to their cognate binding sites. Recent studies have identified several TF-nucleosome interaction modes including end binding (EB), oriented binding, periodic binding, dyad binding, groove binding, and gyre spanning. However, there are substantial experimental challenges in measuring nucleosome binding modes for thousands of TFs in different species.ResultsWe present a computational prediction of the binding modes based on TF protein sequences. With a nested cross-validation procedure, our model outperforms several fine-tuned off-the-shelf machine learning (ML) methods in the multi-label classification task. Our binary classifier for the EB mode performs better than these ML methods with the area under precision-recall curve achieving 75%. The end preference of most TFs is consistent with low nucleosome occupancy around their binding site in GM12878 cells. The nucleosome occupancy data is used as an alternative dataset to confirm the superiority of our EB classifier.ConclusionsWe develop the first ML-based approach for efficient and comprehensive analysis of nucleosome binding modes of TFs.

Revealing biophysical properties of KfrA-type proteins as a novel class of cytoskeletal, coiled-coil plasmid-encoded proteins

BackgroundDNA binding KfrA-type proteins of broad-host-range bacterial plasmids belonging to IncP-1 and IncU incompatibility groups are characterized by globular N-terminal head domains and long alpha-helical coiled-coil tails. They have been shown to act as transcriptional auto-regulators.ResultsThis study was focused on two members of the growing family of KfrA-type proteins encoded by the broad-host-range plasmids, R751 of IncP-1β and RA3 of IncU groups. Comparative in vitro and in silico studies on KfrAR751 and KfrARA3 confirmed their similar biophysical properties despite low conservation of the amino acid sequences. They form a wide range of oligomeric forms in vitro and, in the presence of their cognate DNA binding sites, they polymerize into the higher order filaments visualized as “threads” by negative staining electron microscopy. The studies revealed also temperature-dependent changes in the coiled-coil segment of KfrA proteins that is involved in the stabilization of dimers required for DNA interactions.ConclusionKfrAR751 and KfrARA3 are structural homologues. We postulate that KfrA type proteins have moonlighting activity. They not only act as transcriptional auto-regulators but form cytoskeletal structures, which might facilitate plasmid DNA delivery and positioning in the cells before cell division, involving thermal energy.

Cooperative Activity of α4β1 and α4β7 Integrins in Mediating Human B-Cell Lymphoma Adhesion and Chemotaxis on Fibronectin Through Recognition of Multiple Synergizing Binding Sites Within the Central Cell-Binding Domain

AbstractWe have quantitated the relative contributions of the constitutively active α4β1 and α4β7 integrins and the domains embodying their cognate binding sites in mediating human B-cell lymphoma adhesion and chemotaxis on fibronectin. By cooperating, the central cell-binding and IIICS carboxy-terminal domains were entirely responsible for the adhesion activity displayed by fibronectin, and their relative contribution to this process was estimated to be 30% versus 70%. Assessment of the leukocyte-substrate binding strength (ie, dynes/cell) indicated a 10-fold higher avidity of the cell-IIICS domain interaction. The two integrins interchangeably recognized both domains, but differed quantitatively in their participation in the adhesive event, as well as in domain preference. The use of 3Fn (according to the nomenclature proposed by Bork and Koonin [Curr Opin Struct Biol 6:366, 1996] for the type III fibronectin modules) module-specific antibodies and recombinant polypeptides showed that α4 integrins recognized both the RGD sequence (3Fn10) and an apparently novel synergistic site located within the 3Fn8 module; even in this case, the integrins displayed a distinct binding site preference. Interleukin-1β (IL-1β)/IL-2–induced chemotaxis also involved cooperative function of the central cell-binding and IIICS domains, but the mechanisms regulating this phenomenon differed markedly from those controlling cell adhesion. First, the relative contribution of the individual domains was comparable, but neither of the individual domains promoted migration to the extent observed on intact fibronectin. Secondly, α4β1 and α4β7 integrins were both involved in the domain-binding necessary for initiation of migration, but the relative contribution of each receptor in the chemotactic process was less disparate than for initial cell adhesion. Thirdly, the mode by which chemotactic B-lymphoma movement was supported by the central cell-binding domain differed from that sustaining cell adhesion in that it involved independent recognition of either the 3Fn8 or the 3Fn9 module, which acted in synergy with the 3Fn10 module. Our data provide novel evidence concerning the relative importance of the constitutively active α4β1 and α4β7 integrins for the interaction of B-cell lymphoma cells with fibronectin, and they emphasize a multiple and diverse recognition of sites responsible for either anchorage or locomotion of tumor leukocytes on this matrix molecule.