Functional studies of the ‘GAFTGA’ motif of Escherichia coli Phage Shock Protein F

In the archetypal bacterial transcription, the multi-subunit core RNA polymerase (RNAP) is specifically bound to the promoter through the sigma factor (σ), forming a closed complex where DNA remains double-stranded. The promoter DNA is initially spontaneously melted by the σ factor within the core enzyme and subsequently loaded into the active channel of the holoenzyme. However, a major alternative transcription paradigm that depends on the sigma factor σ54 also exists in bacteria and controls pathogenicity, biofilm formation, bioluminescence, nitrogen fixation and stress responses. σ54 forms inhibitory interactions with DNA in the closed complex, which can only be alleviated by ATP hydrolysis-driven remodelling catalysed by bacterial enhancer binding proteins (bEBPs). In this regard, σ54- dependent transcription can be viewed analogous to the eukaryotic RNA Pol II system. This study was initiated to advance the understanding of: (i) how the ATP hydrolysis energy is relayed from the ATPase catalytic site to the closed complex for remodelling, (ii) the hexameric interface organisation of bEBPs for ATP hydrolysis, and (iii) the role of core RNAP in σ54-dependent transcription. A newly devised cross-linking technique combined with the DNA footprinting methods provided new insights of the organisation of each transcription component. The data gathered from this study updated the current working model for ATPdependent transcription. In addition, the cross-linking method proved to be an excellent tool to study protein-protein and nucleo-protein interactions

Luminescent detection of DNA-binding proteins

Transcription factors play a central role in cell development, differentiation and growth in biological systems due to their ability to regulate gene expression by binding to specific DNA sequences within the nucleus. The dysregulation of transcription factor signaling has been implicated in the pathogenesis of a number of cancers, developmental disorders, inflammation and autoimmunity. There is thus a high demand for convenient high-throughput methodologies able to detect sequence-specific DNA-binding proteins and monitor their DNA-binding activities. Traditional approaches for protein detection include gel mobility shift assays, DNA footprinting and enzyme-linked immunosorbent assays (ELISAs) which tend to be tedious, time-consuming, and may necessitate the use of radiographic labeling. By contrast, luminescence technologies offer the potential for rapid, sensitive and low-cost detection that are amenable to high-throughput and real-time analysis. The discoveries of molecular beacons and aptamers have spearheaded the development of new luminescent methodologies for the detection of proteins over the last decade. We survey here recent advances in the development of luminescent detection methods for DNA-binding proteins, including those based on molecular beacons, aptamer beacons, label-free techniques and exonuclease protection

Prediction of DtxR regulon: Identification of binding sites and operons controlled by Diphtheria toxin repressor in Corynebacterium diphtheriae

BACKGROUND: The diphtheria toxin repressor, DtxR, of Corynebacterium diphtheriae has been shown to be an iron-activated transcription regulator that controls not only the expression of diphtheria toxin but also of iron uptake genes. This study aims to identify putative binding sites and operons controlled by DtxR to understand the role of DtxR in patho-physiology of Corynebacterium diphtheriae. RESULT: Positional Shannon relative entropy method was used to build the DtxR-binding site recognition profile and the later was used to identify putative regulatory sites of DtxR within C. diphtheriae genome. In addition, DtxR-regulated operons were also identified taking into account the predicted DtxR regulatory sites and genome annotation. Few of the predicted motifs were experimentally validated by electrophoretic mobility shift assay. The analysis identifies motifs upstream to the novel iron-regulated genes that code for Formamidopyrimidine-DNA glycosylase (FpG), an enzyme involved in DNA-repair and starvation inducible DNA-binding protein (Dps) which is involved in iron storage and oxidative stress defense. In addition, we have found the DtxR motifs upstream to the genes that code for sortase which catalyzes anchoring of host-interacting proteins to the cell wall of pathogenic bacteria and the proteins of secretory system which could be involved in translocation of various iron-regulated virulence factors including diphtheria toxin. CONCLUSIONS: We have used an in silico approach to identify the putative binding sites and genes controlled by DtxR in Corynebacterium diphtheriae. Our analysis shows that DtxR could provide a molecular link between Fe(+2)-induced Fenton's reaction and protection of DNA from oxidative damage. DtxR-regulated Dps prevents lethal combination of Fe(+2 )and H(2)O(2 )and also protects DNA by nonspecific DNA-binding. In addition DtxR could play an important role in host interaction and virulence by regulating the levels of sortase, a potential vaccine candidate and proteins of secretory system

Information-driven protein–DNA docking using HADDOCK: it is a matter of flexibility

Intrinsic flexibility of DNA has hampered the development of efficient protein−DNA docking methods. In this study we extend HADDOCK (High Ambiguity Driven DOCKing) [C. Dominguez, R. Boelens and A. M. J. J. Bonvin (2003) J. Am. Chem. Soc. 125, 1731–1737] to explicitly deal with DNA flexibility. HADDOCK uses non-structural experimental data to drive the docking during a rigid-body energy minimization, and semi-flexible and water refinement stages. The latter allow for flexibility of all DNA nucleotides and the residues of the protein at the predicted interface. We evaluated our approach on the monomeric repressor−DNA complexes formed by bacteriophage 434 Cro, the Escherichia coli Lac headpiece and bacteriophage P22 Arc. Starting from unbound proteins and canonical B-DNA we correctly predict the correct spatial disposition of the complexes and the specific conformation of the DNA in the published complexes. This information is subsequently used to generate a library of pre-bent and twisted DNA structures that served as input for a second docking round. The resulting top ranking solutions exhibit high similarity to the published complexes in terms of root mean square deviations, intermolecular contacts and DNA conformation. Our two-stage docking method is thus able to successfully predict protein−DNA complexes from unbound constituents using non-structural experimental data to drive the docking

Evidence of the formation of G-quadruplex structures in the promoter region of the human vascular endothelial growth factor gene

The polypurine/polypyrimidine (pPu/pPy) tract of the human vascular endothelial growth factor (VEGF) gene is proposed to be structurally dynamic and to have potential to adopt non-B DNA structures. In the present study, we further provide evidence for the existence of the G-quadruplex structure within this tract both in vitro and in vivo using the dimethyl sulfate (DMS) footprinting technique and nucleolin as a structural probe specifically recognizing G-quadruplex structures. We observed that the overall reactivity of the guanine residues within this tract toward DMS was significantly reduced compared with other guanine residues of the flanking regions in both in vitro and in vivo footprinting experiments. We also demonstrated that nucleolin, which is known to bind to G-quadruplex structures, is able to bind specifically to the G-rich sequence of this region in negatively supercoiled DNA. Our chromatin immunoprecipitation analysis further revealed binding of nucleolin to the promoter region of the VEGF gene in vivo. Taken together, our results are in agreement with our hypothesis that secondary DNA structures, such as G-quadruplexes, can be formed in supercoiled duplex DNA and DNA in chromatin in vivo under physiological conditions similar to those formed in single-stranded DNA templates

Site-specific interactions of JBP with base and sugar moieties in duplex J-DNA - Evidence for both major and minor groove contacts

Bio-organic Synthesi

IDENTIFICATION OF NOVEL GENES REGULATING ELASTIC FIBER FORMATION THROUGH EXPRESSION PROFILING ANALYSIS OF ELASTOGENIC MODELS

Background: Particularly important to the mechanical performance of native arterial blood vessels is elastin, an extracellular matrix (ECM) protein deposited by VSMCs in the form of elastic fibers, arranged in concentric lamellae in the media of the vessel wall. In addition to serving as major structural elements of arterial walls, providing extensibility and elastic recoil, elastic fibers also influence vascular cell behaviors. For these reasons tissue engineers are attempting to exploit elastic fiber biology to enhance vascular graft design and patency. Therefore, developing a greater understanding of the molecular mechanisms of elastogenesis may offer opportunities to control elastogenesis in tissue biofabrication. Approach: To discover genes critical for elastogenesis we performed analysis of gene expression profiles associated with elastogenesis occurring 1) during lung and aorta development, 2) in the lung and skin in response to injury, and 3) in vascular smooth muscle cells (VSMCs) stimulated to produce elastic fibers. On the resulting convergent gene set we employed Promoter Analysis and Interaction Network Toolset (PAINT) to identify transcription factor binding regions. We also mapped binding sites for microRNA (miRNA) within the convergent gene set. Subsequent screening for potential regulators of elastogenesis were performed using pharmacological agonists and antagonists along with plasmid vector transfection to augment expression. Differences in elastin transcription were measured by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and by anti-elastin immunostaining in the development of a novel elastin ELISA. A 3D proof of concept tissue culture model containing fibroblasts and macroporous gelatin microcarrier beads was also established and immunostained for elastin. Results: Our transcriptomic studies revealed a set of genes differentially regulated in all five models of elastogenesis tested. Aside from genes that have previously been established to act in the elastogenesis process there are \u3e50 genes that have not been implicated in elastogenesis. Moreover, promoter analysis of clusters of genes from the 63-gene set having a similar pattern of regulation during developmental elastogenesis revealed two potential elastogenesis regulatory network of TFs. We hypothesize that these sets of genes contain novel positive and negative effectors of elastogenesis. Effects of agonists, antagonists, and expression vectors of these genes on elastin expression were quantified in cultured fibroblasts to identify agents that can be employed to accelerate elastogenesis during tissue biofabrication. Conclusions: The findings highlight a group of genes whose expression is differentially expressed in multiple models of elastin formation and many not previously associated with elastogenesis and thus may represent novel components of elastogenesis. Transcriptional regulatory network analysis revealed potential transcription factor regulators of elastogenesis. Candidate genes and transcription factors were regulated through agonist and antagonist treatment and transfection of plasmid expression vectors in order to augment elastogenesis in vascular tissue biofabrication

Investigation of Genomic Instability Induced by G-Quadruplexes in the Absence of Functional Topoisomerase 1 in Yeast

Topoisomerase 1 (Top1) is an enzyme that removes transcriptionally generated negative supercoils by binding and nicking DNA. Since transcription of guanine-rich DNA leads to the formation of G-quadruplex (G4) structures, Top1’s function likely suppresses G4-formation. In support of this, Top1 significantly reduces co-transcriptional G4 DNA-associated genomic instability at a model G4-motif in Saccharomyces cerevisiae. However, whether Top1 suppresses G4-formation on a genome-wide scale in yeast remains unexplored. Therefore, I aimed to uncover if deletion of Top1 enhances genome-wide G4-formation in S. cerevisiae. As an approach to quantify global G4-formation, I expressed the G4-specific antibody BG4 from a yeast vector to perform chromatin immunoprecipitation next generation sequencing (ChIP-seq) and immunofluorescence experiments. While the G4-antibody’s function was verified in vitro, ChIP and immunofluorescence experiments failed, possibly due to localization of BG4 to the cytoplasm rather than the nucleus of yeast cells. Thus, future attempts at enumerating G4s in TOP1-deletion yeast cells should include the usage of expressed BG4 fused to a nuclear localization signal sequence or purified BG4 protein. Top1 mutants arise in cancer cells treated with the Top1-targeting anticancer drug camptothecin (CPT). Here, I show that the impact on G4-induced recombination in yeast depends on the type of CPT-resistant Top1 mutant expressed. While expression of a Top1 mutant defective in duplex DNA binding results in G4-recombination levels equivalent to cells completely lacking Top1, expression of cleavage-defective Top1 mutants has an even greater impact on G4-mediated instability. I also find that Top1 cleavage-defective mutants bind G4s in vitro and that the SPRTN homolog Wss1 involved in DNA/protein crosslink resolution partly suppresses G4-induced recombination in yeast cells expressing Top1 cleavage-defective mutants. Collectively, these data suggest that Top1 cleavage-defective mutants induce instability at guanine-rich DNA through G4-stabilization in vivo. Further, I uncovered that another G4-binding protein, Nsr1 or yeast nucleolin, contributes to G4-instability in yeast cells expressing Top1 mutants and provide additional evidence indicating that Top1 cleavage-defective mutants and Nsr1 interact when bound to G4s to form a potential replication block. Bioinformatic data revealed that cancer genomes harboring Top1 mutants predicted to be functionally defective exhibit enriched mutagenesis at G4-motifs. Yeast genetic datum showing that Top1 cleavage-defective mutants and Nsr1 have a synergistic effect on G4-instability through cooperative G4-binding taken together with the result of bioinformatic analyses suggest that CPT-resistance conferring Top1 mutants could induce mutagenesis at G4-motifs in cancer cells and complicate patient treatment. Since loss of Top1 function increases G4-instability, identifying additional protein factors that suppress or instigate G4-mediated DNA damage in the absence of functional Top1 is an attractive future direction of this work

Characterisation of bacteriophage-encoded inhibitors of the bacterial RNA polymerase

RNA polymerase (RNAP) is an essential enzyme which catalyses transcription; a highly regulated process. Bacteriophage are viruses which infect bacteria and as a result have evolved a diverse range of mechanisms to regulate the bacterial RNAP to serve the needs of the virus. T7 Gp2 and Xp10 P7 are two bacteriophage-encoded transcription factors that inhibit the activity of the bacterial RNAP. The aim of this study is to investigate the molecular mechanisms of action of Gp2 and P7. Fluorescence anisotropy experiments proved Gp2 to bind to RNAP, independently of the σ- factor, with a 1:1 stoichiometry and a low nanomolar affinity. In vitro transcription assays demonstrated that a negatively charged strip in Gp2 is the major determinant for its inhibitory activity. Furthermore, it was shown that efficient Gp2-mediated inhibition of RNAP also depends upon the highly negatively charged and flexible σ70 specific domain, R1.1. Gp2 and R1.1 both bind in the downstream-DNA binding channel and exert long-range antagonistic effects on RNAP-promoter DNA interactions around the transcription start site. A systematic mutagenesis screen was used to identify residues in P7 necessary for binding to the RNAP; results were interpreted in the context of a newly resolved NMR structure of P7. Electrophoretic mobility shift assays revealed that P7 ‘traps’ a RNAP-promoter DNA complex en route to the transcriptionally-competent complex. Preliminary results from a fluorescence based RNAP-DNA interaction assay suggest that P7 may target RNAP interactions with the -35 promoter element and the ‘discriminator region’. This study has contributed to our understanding of how non-bacterial transcriptional factors can influence bacterial gene expression by modulating RNAP activity. This study has also uncovered vulnerabilities in RNAP, which have the potential to be exploited therapeutically. To this end, these structure-function studies of Gp2 and P7 have provided the basis for the rational design of novel anti-bacterial compounds

Stress induced transcriptional regulation of the glycine transporter type 1A (GlyT-1A/SLC6A9) in human intestinal epithelia

Phd ThesisThere is mounting experimental evidence demonstrating protection by free glycine against stress in several cell types. The glycine transporter type 1 (GlyT-1) mediates the high affinity supply of glycine, which together with cysteine is required for the synthesis of the antioxidant glutathione. Previous work in this laboratory has established that GlyT-1 is expressed on the apical and basal membranes of intestinal epithelial cells and that its mRNA levels are regulated by stress. In the present study exactly how stress signals to transcriptional induction of GlyT-1 was investigated. Caco-2 cells transfected with reporter constructs of sequences of the GlyT-1a proximal promoter and 5’UTR cloned upstream of a β-galactosidase coding sequence, showed increased reporter activity following treatment with thapsigargin (Tg), tunicamycin (Tu), amino acid (AA) starvation, tert-Butylhydroquinone (tBHQ) or Diethyl maleate (DEM). Despite no changes in Nrf-2 mRNA levels, a significant increase in total Nrf-2 protein abundance was evident on western-blots following DEM treatment of Caco-2 cells. However, gel shift showed no protein-DNA complexes between Nrf-2 protein and a DNA probe sequence of the putative antioxidant response element (ARE) identified in the GlyT-1a 5’ flank. Despite a significant siRNA mediated knock-down of Nrf-2 mRNA and protein, there was no further effect on GlyT-1a expression. Unlike Nrf-2, the knock-down of Atf-4 diminished the basal and stressed induced expression of GlyT-1a. Atf-4 was detected bound to DNA probes containing a potential amino acid response element (AARE) located in the first exon of the GlyT-1a gene by gel shift and super shift assays. QPCR assays performed on DNA isolated from Caco-2 cells by chromatin immunoprecipitation (ChIP) using antibodies against Atf-4, demonstrated 9, 5 and 2-fold enrichment of the GlyT-1a AARE following Tu, AA starvation and DEM treatment respectively. Site directed mutation of the GlyT-1a AARE showed a 75% reduction in reporter activity as well as attenuated protein-DNA interaction with a representative probe. It is evident from the data presented in this thesis that the direct interaction of Atf-4 at the proposed GlyT-1a AARE contributes to its transcriptional up-regulation following endoplasmic reticulum stress, nutrient stress and oxidative stress