Biomedical subjects

Tomasz Heyduk

Publications and source records attributed to Tomasz Heyduk.

At least 19 recordsLinked to original sources

PubMed2006

A basal promoter element recognized by free RNA polymerase sigma subunit determines promoter recognition by RNA polymerase holoenzyme.

During transcription initiation by bacterial RNA polymerase, the sigma subunit recognizes the -35 and -10 promoter elements; free sigma, however, does not bind DNA. We selected ssDNA aptamers that strongly and specifically bound free sigma(A) from Thermus aquaticus. A consensus sequence, GTA(C/T)AATGGGA, was required for aptamer binding to sigma(A), with the TA(C/T)AAT segment making interactions similar to those made by the -10 promoter element (consensus sequence TATAAT) in the context of RNA polymerase holoenzyme. When in dsDNA form, the aptamers function as strong promoters for the T. aquaticus RNA polymerase sigma(A) holoenzyme. Recognition of the aptamer-based promoters depends on the downstream GGGA motif from the aptamers' common sequence, which is contacted by sigma(A) region 1.2 and directs transcription initiation even in the absence of the -35 promoter element. Thus, recognition of bacterial promoters is controlled by independent interactions of sigma with multiple basal promoter elements.

Aptamers, Nucleotide↗

PubMed2006

A consensus adenine at position -11 of the nontemplate strand of bacterial promoter is important for nucleation of promoter melting.

Numerous studies have suggested an important role of adenine at position -11 of the nontemplate strand of bacterial promoters for sequence-specific recognition of the -10 promoter element in single-stranded form. In this work, we attempted to identify a specific step in transcription initiation reaction that is most critically dependent on specific recognition of -11A. Mutating -11A in the context of a model promoter resulted in a profound decrease of the rate of heparin-resistant promoter complex formation and in a modest increase of the rate of heparin-resistant complex dissociation. The identity of nontemplate base at position -11 became relatively unimportant when the duplex in the vicinity of this position was destabilized by base pair mismatches. For promoters with a nonnative thymine at nontemplate position -11, we observed a remarkable correlation between the rate of heparin-resistant complex formation (or transcription activity) and the free energy of duplex stability in the vicinity of this residue, indicating that the replacement of -11A with a T affected a step in the reaction that involves local melting of DNA duplex. These data show that a promoter melting defect caused by a loss of RNA polymerase contact with -11A can be rescued by artificially induced local destabilization of the DNA duplex. These results are consistent with and support the idea that specific recognition of adenine at the nontemplate -11-position is important only for the initial nucleation of melting, which probably involves the flipping of this adenine out from the DNA duplex.

Adenine↗

PubMed2005

Transcription regulation of the EcoRV restriction-modification system.

When a plasmid containing restriction-modification (R-M) genes enters a naïve host, unmodified host DNA can be destroyed by restriction endonuclease. Therefore, expression of R-M genes must be regulated to ensure that enough methyltransferase is produced and that host DNA is methylated before the endonuclease synthesis begins. In several R-M systems, specialized Control (C) proteins coordinate expression of the R and the M genes. C proteins bind to DNA sequences called C-boxes and activate expression of their cognate R genes and inhibit the M gene expression, however the mechanisms remain undefined. Here, we studied the regulation of gene expression in the C protein-dependent EcoRV system. We map the divergent EcoRV M and R gene promoters and we define the site of C protein-binding that is sufficient for activation of the EcoRV R transcription.

Bacterial Proteins↗

PubMed2005

Synaptic complex formation of two retrovirus DNA attachment sites by integrase: a fluorescence energy transfer study.

The integration of retroviral DNA by the viral integrase (IN) into the host genome occurs via assembled preintegration complexes (PIC). We investigated this assembly process using purified IN and viral DNA oligodeoxynucleotide (ODN) substrates (93 bp in length) that were labeled with donor (Cy3) and acceptor fluorophores (Cy5). The fluorophores were attached to the 5' 2 bp overhangs of the terminal attachment (att) sites recognized by IN. Addition of IN to the assay mixture containing the fluorophore-labeled ODN resulted in synaptic complex formation at 14 degrees C with significant fluorescence resonance energy transfer (FRET) occurring between the fluorophores in close juxtaposition (from approximately 15 to 100 A). Subsequent integration assays at 37 degrees C with the same ODN (32P-labeled) demonstrated a direct association of a significant FRET signal with concerted insertion of the two ODNs into the circular DNA target, here termed full-site integration. FRET measurements (deltaF) show that IN binds to a particular set of 3' OH recessed substrates (type I) generating synaptic complexes capable of full-site integration that, as shown previously, exhibit IN mediated protection from DNaseI digestion up to approximately 20 bp from the ODN att ends. In contrast, IN also formed complexes with nonspecific DNA ends and loss-of-function att end substrates (type II) that had significantly lower deltaF values and were not capable of full-site integration, and lacked the DNaseI protection properties. The type II category may exemplify what is commonly understood as "nonspecific" binding by IN to DNA ends. Two IN mutants that exhibited little or no integration activity gave rise to the lower deltaF signals. Our FRET analysis provided the first direct physical evidence that IN forms synaptic complexes with two DNA att sites in vitro, yielding a complex that exhibits properties comparable to that of the PIC.

Attachment Sites, Microbiological↗

PubMed2005

Escherichia coli RNA polymerase contacts outside the -10 promoter element are not essential for promoter melting.

We examined the relative affinity of model promoter constructs for binding Escherichia coli RNA polymerase (RNAP) holoenzyme. Model promoter constructs were designed to mimic DNA structures characteristic for different steps of transcription initiation. DNA duplexes in which a chemical cross-link was introduced just downstream from -10 hexamer to prevent DNA melting upon RNAP binding were used to mimic RNAP-promoter contacts in a closed complex. Fork junction DNA molecules with double-stranded/single-stranded junction between -11 and -10 position were used to study interactions of RNA polymerase with DNA in open complex. The -35 and -10 promoter regions were found to be equally important for the initial RNAP binding. The recognition of -35 promoter region was independent of structural context of the model promoter fragment. In contrast, free energy of RNAP binding to -10 hexamer was highly dependent on DNA structure. The relative importance of -10 region for sequence-specific interaction with the polymerase was the lowest for constructs mimicking closed complex and the highest for the constructs mimicking open complex. The relative importance of region -10 was also dependent on the presence of -35 consensus element indicating a communication between different DNA binding determinants of polymerase during open complex formation. Short double-stranded promoter fragments comprising only -35 and -10 or only -10 consensus elements underwent melting in a complex with polymerase indicating that the core of promoter melting activity of the polymerase is localized to a very small subset of all promoter-polymerase contacts.

Binding, Competitive↗

PubMed2005

Nucleic acid-based fluorescence sensors for detecting proteins.

We report here development of a rapid, homogeneous, aptamer-based fluorescence assay ("molecular beacons") for detecting proteins. The assay involves protein-induced coassociation of two aptamers recognizing two distinct epitopes of the protein. The aptamers contain short fluorophore-labeled complementary "signaling" oligonucleotides attached to the aptamer by non-DNA linker. Coassociation of the two aptamers with the protein results in bringing the two "signaling" oligonucleotides into proximity, producing a large change of fluorescence resonance energy transfer between the fluorophores. We used thrombin as a model system to provide proof-of-principle evidence validating this molecular beacon design. Thrombin beacon was capable of detecting the protein with high selectivity (also in complex biological mixtures), picomolar sensitivity, and high signal-to-background ratio. This is a homogeneous assay requiring no sample manipulation. Since the design of molecular beacons described here is not limited to any specific protein, it will be possible to develop these beacons to detect a variety of target proteins of biomedical importance.

Biosensing Techniques↗

PubMed2004

Unimolecular beacons for the detection of DNA-binding proteins.

A new methodology for detecting sequence-specific DNA-binding proteins has been recently developed (Heyduk, T.; Heyduk, E. Nat. Biotechnol. 2002, 20, 171). The core feature of this methodology is protein-dependent association of two fluorochrome-labeled DNA fragments, which allows generation of a fluorescence signal reporting the presence of the target protein. Previous kinetic experiments identified the association of the two DNA fragments as the rate-limiting step of the assay. Here we report on a variant of the assay, in which components of the assay--fluorescent DNA fragments--were covalently tethered by a non-DNA linker with the goal of increasing the rate of association of the two fragments. We investigated the effect of the tether on the performance of the assay under a variety of conditions using a model DNA-binding protein. Quantitative titrations and rapid kinetic stopped-flow experiments were conducted to validate the molecular model that describes the two linked equilibria: oscillation of the tethered construct between the open and closed states and the exclusive association of the protein with the closed state. Experiments were also performed to demonstrate the ability of these tethered constructs to signal when attached to a solid surface. The major advantage of this new assay format is the faster response time for the detection allowing the higher throughput of the analysis. Additionally, it will be possible to attach tethered beacons to other solid surfaces, thus allowing the preparation of arrays containing molecular beacons for many different DNA-binding proteins.

DNA↗

PubMed2004

Zinc inhibition of adenylyl cyclase correlates with conformational changes in the enzyme.

We have previously demonstrated that Zn(2+) inhibits hormone and forskolin stimulation of cAMP synthesis in intact N18TG2 cells, corresponding plasma membranes, and of recombinant adenylyl cyclase isoforms. If, however, the enzyme is pre-activated by hormone or forskolin, Zn(2+) inhibition is attenuated [J. Biol. Chem. 277 (2002) 11859]. We have extended our analyses of this inhibition to investigations of soluble adenylyl cyclase, composed of the CI and CII domains of the full-length protein. The properties of Zn(2+) inhibition of the soluble enzyme parallel that of the full-length protein, including the fact that inhibition is not competitive with Mg(2+). By monitoring intrinsic and extrinsic fluorescence, we demonstrate changes in enzyme conformers in response to the addition of varied effectors. The data suggest a possible mechanism by which Zn(2+) inhibits adenylyl cyclase activity.

Adenylyl Cyclases↗

PubMed2003

High mobility group A2 protein and its derivatives bind a specific region of the promoter of DNA repair gene ERCC1 and modulate its activity.

High mobility group A2 (HMGA2) chromosomal non-histone protein and its derivatives play an important role in development and progression of benign and malignant tumors, obesity and arteriosclerosis, although the underlying mechanisms of these conditions are poorly understood. Therefore, we tried to identify target genes for this transcriptional regulator and to provide insights in the mechanism of interaction to its target. Multiple genes have been identified by microarray experiments as being transcriptionally regulated by HMGA2. Among these we chose the ERCC1 gene, encoding a DNA repair protein, for this study. DNA-binding studies were performed using HMGA2 and C-terminally truncated DeltaHMGA2, a derivative that is frequently observed in a variety of tumors. A unique high affinity HMGA2 binding site was mapped to a specific AT-rich region located -323 to -298 upstream of the ERCC1 transcription start site, distinguishing it from other potential AT-rich binding sites. The observed 1:1 stoichiometry for the binding of wild-type HMGA2 to this region was altered to 1:2 upon binding of truncated DeltaHMGA2, causing DNA bending. Furthermore, the regulatory effect of HMGA2 was confirmed by luciferase promoter assays showing that ERCC1 promoter activity is down-regulated by all investigated HMGA2 forms, with the most striking effect exerted by DeltaHMGA2. Our results provide the first insights into how HMGA2 and its aberrant forms bind and regulate the ERCC1 promoter.

Binding Sites↗

PubMed2003

Characterization of the interactions between the bacteriophage T4 AsiA protein and RNA polymerase.

The anti-sigma factor AsiA effects a change in promoter specificity of the Escherichia coli RNA polymerase via interactions with two conserved regions of the sigma(70) subunit, denoted 4.1 and 4.2. Free AsiA is a symmetrical homodimer. Here, we show that AsiA is monomeric when bound to sigma(70) and that a subset of the residues that contribute to the homodimer interface also contributes to the interface with sigma(70). AsiA interacts primarily with C-terminal sections of regions 4.1 and 4.2, which show remarkable sequence similarity. An AsiA monomer can simultaneously, and apparently cooperatively, bind both isolated regions 4.1 and 4.2 at preferred, distinct subsites, whereas region 4.1 alone or region 4.2 alone can interact with either subsite. These results suggest structural and functional plasticity in the interaction of AsiA with sigma(70) and support the notion of discrete roles for regions 4.1 and 4.2 in transcription regulation by AsiA. Furthermore, we show that AsiA inhibits recognition of the -35 consensus promoter element by region 4 of sigma(70) indirectly, as the residues on region 4 responsible for AsiA binding are distinct from those involved in DNA binding. Finally, we show that AsiA must directly disrupt the interaction of region 4 with the RNA polymerase beta subunit flap domain, resulting in a distance change between region 2 and region 4 of sigma(70). Thus, a new paradigm for transcription regulation by AsiA is emerging, whereby the distance between the DNA binding domains in sigma(70) is regulated, and promoter recognition specificity is modulated, by mediating the interactions of the sigma region 4 with the beta subunit flap domain.

Bacteriophage T4↗

PubMed2003

Molecular beacons for detecting DNA binding proteins: mechanism of action.

New methodology for detecting sequence-specific DNA binding proteins has been recently developed (T. Heyduk, and E. Heyduk, Nat. Biotechnol. 20 (2002) 171). The central feature of this assay is protein-dependent association of two DNA fragments, each containing about half of a DNA sequence-defining the protein binding site. In this report we propose a physical model explaining the functioning of the assay. The model involves two linked equilibria: association between the two DNA fragments and binding of the protein exclusively to the complex between the two DNA fragments. Equilibrium and kinetic experiments provided evidence supporting the proposed model and showed that the model was sufficient to describe the behavior of the assay under a variety of conditions. Kinetic data identified the association between the two DNA half-sites as the rate-limiting step of the assay. Theoretical simulations based on the proposed model were used to investigate parameters important for the maximal sensitivity of the assay. Physical understanding of the assay will provide means for rational design of the assay for a variety of target proteins.

Binding Sites↗

PubMed2003

Interaction of T4 AsiA with its target sites in the RNA polymerase sigma70 subunit leads to distinct and opposite effects on transcription.

Bacteriophage T4 AsiA is a homodimeric protein that orchestrates a switch from the host and early viral transcription to middle viral transcription by binding to the sigma(70) subunit of Escherichia coli RNA polymerase holoenzyme (Esigma(70)) and preventing promoter complex formation on most E.coli and early T4 promoters. In addition, Esigma(70)AsiA, but not Esigma(70), is a substrate of transcription activation by T4-encoded DNA-binding protein MotA, a co-activator of transcription from middle viral promoters. The molecular determinants of sigma(70)-AsiA interaction necessary for transcription inhibition reside in the sigma(70) conserved region 4.2, which recognizes the -35 promoter consensus element. The molecular determinants of sigma(70)-AsiA interaction necessary for MotA-dependent transcription activation have not been identified. Here, we show that in the absence of sigma(70) region 4.2, AsiA interacts with sigma(70) conserved region 4.1 and activates transcription in a MotA-independent manner. Further, we show that the AsiA dimer must dissociate to interact with either region 4.2 or region 4.1 of sigma(70). We propose that MotA may co-activate transcription by restricting AsiA binding to sigma(70) region 4.1.

Amino Acid Sequence↗

PubMed2003

Luminescence resonance energy transfer-based high-throughput screening assay for inhibitors of essential protein-protein interactions in bacterial RNA polymerase.

The binding of sigma factors to core RNA polymerase is essential for the specific initiation of transcription in eubacteria and is thus critical for cell growth. Since the responsible protein-binding regions are highly conserved among all eubacteria but differ significantly from eukaryotic RNA polymerases, sigma factor binding is a promising target for drug discovery. A homogeneous assay for sigma binding to RNA polymerase (Escherichia coli) based on luminescence resonance energy transfer (LRET) was developed by using a europium-labeled sigma70 and an IC5-labeled fragment of the beta' subunit of RNA polymerase (amino acid residues 100 through 309). Inhibition of sigma binding was measured by the loss of LRET through a decrease in IC5 emission. The technical advances offered by LRET resulted in a very robust assay suitable for high-throughput screening, and LRET was successfully used to screen a crude natural-product library. We illustrate this method as a powerful tool to investigate any essential protein-protein interaction for basic research and drug discovery.

Animals↗

PubMed2003

Homogeneous fluorescence assay for cyclic AMP.

The objective of these studies was to develop a new homogeneous fluorescence assay for determining the concentrations of cAMP in biological samples. The assay is based on a novel general concept of using ligand-dependent sequence-specific DNA binding proteins as sensors for their respective ligands. CAP protein, a bacterial DNA binding protein whose DNA binding activity depends on cAMP, was used to develop the assay. In the presence of cAMP, DNA binding activity of CAP is greatly increased. Signaling of cAMP presence was achieved by detecting cAMP-dependent formation of CAP-DNA complex using a recently developed fluorescence assay for DNA binding proteins (Heyduk, T., and Heyduk, E. Nature Biotechnology 20,171-176, 2002). Both 96-well and 384-well black microplate formats of the assay were developed and used to detect cAMP in low nanomolar concentrations. The assay involves mixing of the sample with the assay solution containing all necessary components for cAMP determination followed by fluorescence intensity readout; no washing or reagent addition steps are necessary. Excellent reproducibility of fluorescence signal change as a function of cAMP concentration was observed. Experiments with HEK 293 cells stimulated with forskolin were performed to demonstrate that the assay could be used for cAMP determination in cellular extracts. In summary, the obtained data fully validated the new homogenous assay for measuring cAMP based on cAMP-dependent DNA binding activity of CAP protein. It is expected that the development of assays for many other ligands of DNA binding proteins will be possible using the same overall assay design developed for cAMP.

Cells, Cultured↗

PubMed2002

On-column tris(2-carboxyethyl)phosphine reduction and IC5-maleimide labeling during purification of a RpoC fragment on a nickel-nitrilotriacetic acid Column.

Fluorescence labeling of proteins has become increasingly important since fluorescent techniques like FRET and fluorescence polarization are now commonly used in protein binding studies, proteomics, and for high-throughput screening in drug discovery. In our efforts to study the binding of the beta(')-subunit from Escherichia coli RNA polymerase (RNAP) to sigma70, we synthesized a fluorescent-labeled beta(')-fragment (residues 100-309) in a very convenient way, that could be used as a general protocol for hexahistidine-tagged proteins. By performing all the following steps, purification, reduction, derivatization with IC5-maleimide, and free dye removal while the protein was bound to the column, we were able to reduce the procedure time significantly and at the same time achieve better labeling efficiency and quality. The beta(')-fragment with a N-terminal His(6)-tag was purified from inclusion bodies and could be refolded prior to or after binding to a Ni-NTA affinity column. Reduction prior to labeling was achieved with TCEP that does not interfere with Ni-NTA chemistry. The labeled beta(')-fragment was tested with sigma70 that was labeled with an europium-based fluorophore for binding in a electrophoretic mobility-shift assay. The sigma-to-core protein interaction in bacterial RNA polymerase offers a potentially specific target for drug discovery, since it is highly conserved among the eubacteria, but differs significantly from eukaryotes.

DNA-Directed RNA Polymerases↗

PubMed2002

A novel bacteriophage-encoded RNA polymerase binding protein inhibits transcription initiation and abolishes transcription termination by host RNA polymerase.

Xp10 is a lytic bacteriophage of Xanthomonas oryzae, a Gram-negative bacterium that causes rice blight. We purified an Xp10 protein, p7, that binds to and inhibits X. oryzae RNA polymerase (RNAP). P7 is a novel 73 amino acid-long protein; it does not bind to and hence does not affect transcription by Escherichia coli RNAP. Analysis of E. coli/X. oryzae RNAP hybrids locates the p7 binding site to the largest X. oryzae RNAP subunit, beta'. Binding of p7 to X. oryzae RNAP holoenzyme prevents large conformational change that places the sigma subunit region 4 into the correct position for interaction with the -35 promoter element. As a result, open promoter complex formation on the -10/-35 class promoters is inhibited. Inhibition of promoter complex formation on the extended -10 class promoters is less efficient. The p7 protein also abolishes factor-independent transcription termination by X. oryzae RNAP by preventing the release of nascent RNA at terminators. Further physiological and mechanistic studies of this novel transcription factor should provide additional insights into its biological role and the processes of promoter recognition and transcription termination.

Bacteriophages↗

PubMed2002

Conformation of fork junction DNA in a complex with Escherichia coli RNA polymerase.

Escherichia coli RNA polymerase is able to bind fork junction DNA containing a conserved -10 promoter element in a sequence-specific manner, and it is believed that polymerase-fork junction DNA interaction mimics those between the enzyme and the promoter DNA in the open complex. In this report we determined the conformation of polymerase-bound fork junction DNA in solution. A series of distances between sites in the fork junction DNA in complex with polymerase were determined using luminescence and fluorescence resonance energy transfer. A series of fork junction DNAs were prepared containing the luminescent or fluorescent donor probe at the upstream or at the downstream end of the fork DNA and acceptor probes at nine positions within the fork junction DNA. The measured distances were compared with analogous distances in a model reference DNA duplex, and the observed distance differences were used to build a model of the fork junction DNA in a complex with the polymerase. The obtained model revealed an insignificant perturbation of the duplex part of the fork DNA in a complex with the polymerase whereas a sharp kink of DNA was observed at the ds/ss DNA boundary of the fork junction DNA.

Base Sequence↗

PubMed2002

A role for interaction of the RNA polymerase flap domain with the sigma subunit in promoter recognition.

In bacteria, promoter recognition depends on the RNA polymerase sigma subunit, which combines with the catalytically proficient RNA polymerase core to form the holoenzyme. The major class of bacterial promoters is defined by two conserved elements (the -10 and -35 elements, which are 10 and 35 nucleotides upstream of the initiation point, respectively) that are contacted by sigma in the holoenzyme. We show that recognition of promoters of this class depends on the "flexible flap" domain of the RNA polymerase beta subunit. The flap interacts with conserved region 4 of sigma and triggers a conformational change that moves region 4 into the correct position for interaction with the -35 element. Because the flexible flap is evolutionarily conserved, this domain may facilitate promoter recognition by specificity factors in eukaryotes as well.

Allosteric Regulation↗