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A M Edwards

Publications and source records attributed to A M Edwards.

At least 37 records · Page 2Linked to original sources
PubMed2001

Stimulation of DNA synthesis and c-fos mRNA expression in primary rat hepatocytes by estrogens.

The mechanism(s) of tumour promotion in liver by estrogens is not well understood although growth stimulation is known to be one important element of their action. As a basis for studying mechanisms of growth control by estrogens, effects of both natural and synthetic estrogens on DNA synthesis and protooncogene c-fos mRNA expression were examined in primary cultures of normal rat hepatocytes. 17beta-Estradiol (E(2)) alone was stimulatory and exhibited dramatic synergism with epidermal growth factor (EGF) in stimulating DNA synthesis. All estrogens tested (natural, synthetic, steroidal and non-steroidal) exhibited an ability to stimulate hepatocyte DNA synthesis. This appears to correlate with their ability to induce c-fos mRNA expression. In contrast to a non-estrogenic liver tumour promoter, phenobarbital, insulin is not permissive for the growth-stimulatory action of E(2). Dexamethasone, which is required for stimulation of DNA synthesis by the non-estrogenic tumour promoter alpha-hexachlorocyclohexane and tetradecanoylphorbol acetate, completely blocked E(2)-stimulated DNA synthesis. Such differential requirements for auxiliary factors suggests that estrogen and other non-estrogenic liver tumour promoters act via distinct mechanisms in stimulating hepatocyte DNA synthesis. E(2) alone had no effect, but when in combination with EGF significantly induced c-fos mRNA expression at early times in culture (maximal at 10 h in culture). Such findings, coupled with the observations that (i) E(2) and EGF were synergistic in growth stimulation, (ii) estrogen receptor levels are higher at early times in culture and (iii) the growth-stimulatory ability of E(2) is limited to 4-24 h in culture, support the notion that in hepatocytes E(2) acts via the estrogen receptor to transactivate c-fos expression (an interaction with EGF), which ultimately culminates in enhanced DNA synthesis. Dexamethasone did not block E(2)-induced c-fos gene expression, suggesting that it acts in a pathway(s) distal to activation of fos gene expression. The possible inhibitory mechanisms of action of dexamethasone on E(2)-stimulated DNA synthesis are discussed.

3T3 Cells↗
PubMed2001

Herpes simplex virus triggers and then disarms a host antiviral response.

Virus infection induces an antiviral response that is predominantly associated with the synthesis and secretion of soluble interferon. Here, we report that herpes simplex virus type 1 virions induce an interferon-independent antiviral state in human embryonic lung cells that prevents plaquing of a variety of viruses. Microarray analysis of 19,000 human expressed sequence tags revealed induction of a limited set of host genes, the majority of which are also induced by interferon. Genes implicated in controlling the intracellular spread of virus and eliminating virally infected cells were among those induced. Induction of the cellular response occurred in the absence of de novo cellular protein synthesis and required viral penetration. In addition, this response was only seen when viral gene expression was inhibited, suggesting that a newly synthesized viral protein(s) may function as an inhibitor of this response.

Animals↗
PubMed2000

Dissociable Rpb4-Rpb7 subassembly of rna polymerase II binds to single-strand nucleic acid and mediates a post-recruitment step in transcription initiation.

The Rpb4 and Rpb7 subunits of yeast RNA polymerase II form a heterodimeric complex essential for promoter-directed transcription initiation in a reconstituted system. Results of template competition experiments indicate that the Rpb4-Rpb7 complex is not required for stable recruitment of polymerase to active preinitiation complexes, suggesting that Rpb4-Rpb7 mediates an essential step subsequent to promoter binding. Sequence and structure-based alignments revealed a possible OB-fold single-strand nucleic acid-binding motif in Rpb7. Purified Rpb4-Rpb7 complex exhibited both single-strand DNA- and RNA-binding activities, and a small deletion in the putative OB-fold nucleic acid-binding surface of Rpb7 abolished binding activity without affecting the stability of the Rpb4-Rpb7 complex or its ability to associate with polymerase. The same mutation destroyed the transcription activity of the Rpb4-Rpb7 complex. A separate deletion elsewhere in the OB-fold motif of Rpb7 also blocked transcription but did not affect nucleic acid binding, suggesting that the OB-fold of Rpb7 mediates both DNA-protein and protein-protein interactions required for productive initiation.

Amino Acid Motifs↗
PubMed2000

RNA polymerase II subunit Rpb9 regulates transcription elongation in vivo.

RNA polymerase II lacking the Rpb9 subunit uses alternate transcription initiation sites in vitro and in vivo and is unable to respond to the transcription elongation factor TFIIS in vitro. Here, we show that RPB9 has a synthetic phenotype with the TFIIS gene. Disruption of RPB9 in yeast also resulted in sensitivity to 6-azauracil, which is a phenotype linked to defects in transcription elongation. Expression of the TFIIS gene on a high-copy plasmid partially suppressed the 6-azauracil sensitivity of Deltarpb9 cells. We set out to determine the relevant cellular role of yeast Rpb9 by assessing the ability of 20 different site-directed and deletion mutants of RPB9 to complement the initiation and elongation defects of Deltarpb9 cells in vivo. Rpb9 is composed of two zinc ribbons. The N-terminal zinc ribbon restored the wild-type pattern of initiation start sites, but was unable to complement the growth defects associated with defects in elongation. Most of the site-directed mutants complemented the elongation-specific growth phenotypes and reconstituted the normal pattern of transcription initiation sites. The anti-correlation between the growth defects of cells disrupted for RPB9 and the selection of transcription start sites suggests that this is not the primary cellular role for Rpb9. Genome-wide transcription profiling of Deltarpb9 cells revealed only a few changes, predominantly in genes related to metabolism.

Alanine↗
PubMed2000

Insights into ligand binding and catalysis of a central step in NAD+ synthesis: structures of Methanobacterium thermoautotrophicum NMN adenylyltransferase complexes.

Nicotinamide mononucleotide adenylyltransferase (NMNATase) catalyzes the linking of NMN(+) or NaMN(+) with ATP, which in all organisms is one of the common step in the synthesis of the ubiquitous coenzyme NAD(+), via both de novo and salvage biosynthetic pathways. The structure of Methanobacterium thermoautotrophicum NMNATase determined using multiwavelength anomalous dispersion phasing revealed a nucleotide-binding fold common to nucleotidyltransferase proteins. An NAD(+) molecule and a sulfate ion were bound in the active site allowing the identification of residues involved in product binding. In addition, the role of the conserved (16)HXGH(19) active site motif in catalysis was probed by mutagenic, enzymatic and crystallographic techniques, including the characterization of an NMN(+)/SO4(2-) complex of mutant H19A NMNATase.

Amino Acid Sequence↗
PubMed2000

Structural basis for the recognition of DNA repair proteins UNG2, XPA, and RAD52 by replication factor RPA.

Replication protein A (RPA), the nuclear ssDNA-binding protein in eukaryotes, is essential to DNA replication, recombination, and repair. We have shown that a globular domain at the C terminus of subunit RPA32 contains a specific surface that interacts in a similar manner with the DNA repair enzyme UNG2 and repair factors XPA and RAD52, each of which functions in a different repair pathway. NMR structures of the RPA32 domain, free and in complex with the minimal interaction domain of UNG2, were determined, defining a common structural basis for linking RPA to the nucleotide excision, base excision, and recombinational pathways of repairing damaged DNA. Our findings support a hand-off model for the assembly and coordination of different components of the DNA repair machinery.

Amino Acid Sequence↗
PubMed2000

Structure of a conserved domain common to the transcription factors TFIIS, elongin A, and CRSP70.

TFIIS is a transcription elongation factor that consists of three domains. We have previously solved the structures of domains II and III, which stimulate arrested polymerase II elongation complexes in order to resume transcription. Domain I is conserved in evolution from yeast to human species and is homologous to the transcription factors elongin A and CRSP70. Domain I also interacts with the transcriptionally active RNA polymerase II holoenzyme and therefore, may have a function unrelated to the previously described transcription elongation activity of TFIIS. We have solved the structure of domain I of yeast TFIIS using NMR spectroscopy. Domain I is a compact four-helix bundle that is structurally independent of domains II and III of the TFIIS. Using the yeast structure as a template, we have modeled the homologous domains from elongin A and CRSP70 and identified a conserved positively charged patch on the surface of all three proteins, which may be involved in conserved functional interactions with the transcriptional machinery.

Amino Acid Sequence↗
PubMed2000

Structure-based functional classification of hypothetical protein MTH538 from Methanobacterium thermoautotrophicum.

The structure of MTH538, a previously uncharacterized hypothetical protein from Methanobacterium thermoautotrophicum, has been determined by NMR spectroscopy. MTH538 is one of numerous structural genomics targets selected in a genome-wide survey of uncharacterized sequences from this organism. MTH538 is a so-called singleton, a sequence not closely related to any other (known) sequences. The structure of MTH538 closely resembles the known structures of receiver domains from two component response regulator systems, such as CheY, and is similar to the structures of flavodoxins and GTP-binding proteins. Tests on MTH538 for characteristic activities of CheY and flavodoxin were negative. MTH538 did not become phosphorylated in the presence of acetyl phosphate and Mg(2+), although it appeared to bind Mg(2+). MTH538 also did not bind flavin mononucleotide (FMN) or coenzyme F(420). Nevertheless, sequence and structure parallels between MTH538/CheY and two families of ATPase/phosphatase proteins suggest that MTH538 may have a role in a phosphorylation-independent two-component response regulator system.

Adenosine Triphosphatases↗
PubMed2000

Crystal structure of dTDP-4-keto-6-deoxy-D-hexulose 3,5-epimerase from Methanobacterium thermoautotrophicum complexed with dTDP.

Deoxythymidine diphosphate (dTDP)-4-keto-6-deoxy-d-hexulose 3, 5-epimerase (RmlC) is involved in the biosynthesis of dTDP-l-rhamnose, which is an essential component of the bacterial cell wall. The crystal structure of RmlC from Methanobacterium thermoautotrophicum was determined in the presence and absence of dTDP, a substrate analogue. RmlC is a homodimer comprising a central jelly roll motif, which extends in two directions into longer beta-sheets. Binding of dTDP is stabilized by ionic interactions to the phosphate group and by a combination of ionic and hydrophobic interactions with the base. The active site, which is located in the center of the jelly roll, is formed by residues that are conserved in all known RmlC sequence homologues. The conservation of the active site residues suggests that the mechanism of action is also conserved and that the RmlC structure may be useful in guiding the design of antibacterial drugs.

Amino Acid Sequence↗
PubMed2000

Two domains of the epstein-barr virus origin DNA-binding protein, EBNA1, orchestrate sequence-specific DNA binding.

The EBNA1 (for Epstein-Barr nuclear antigen 1) protein of Epstein-Barr virus governs the replication and partitioning of the viral genomes during latent infection by binding to specific recognition sites in the viral origin of DNA replication. The crystal structure of the DNA binding portion of the EBNA1 protein revealed that this region comprises two structural motifs; a core domain, which mediates protein dimerization and is structurally homologous to the DNA binding domain of the papillomavirus E2 protein, and a flanking domain, which mediated all the observed sequence-specific contacts. To test the possibility that the EBNA1 core domain plays a role in sequence-specific DNA binding not revealed in the crystal structure, we examined the effects of point mutations in potential hydrogen bond donors located in an alpha-helix of the EBNA1 core domain whose structural homologue in E2 mediates sequence-specific DNA binding. We show that these mutations severely reduce the affinity of EBNA1 for its recognition site, and that the core domain, when expressed in the absence of the flanking domain, has sequence-specific DNA binding activity. Flanking domain residues were also found to contribute to the DNA binding activity of EBNA1. Thus, both the core and flanking domains of EBNA1 play direct roles in DNA recognition.

Binding Sites↗
PubMed2000

Solution structure of the RNA polymerase subunit RPB5 from Methanobacterium thermoautotrophicum.

RPB5 is an essential subunit of eukaryotic and archaeal RNA polymerases. It is a proposed target for transcription activator proteins in eukaryotes, but the mechanism of interaction is not known. We have determined the solution structure of the RPB5 subunit from the thermophilic archeon, Methanobacterium thermoautotrophicum. MtRBP5 contains a four-stranded beta-sheet platform supporting two alpha-helices, one on each side of the beta-sheet, resulting in an overall mushroom shape that does not appear to have any structural homologues in the structural database. The position and conservation of charged surface residues suggests possible modes of interaction with other proteins, as well as a rationale for the thermal stability of this protein.

Amino Acid Sequence↗
PubMed2000

Zinc-bundle structure of the essential RNA polymerase subunit RPB10 from Methanobacterium thermoautotrophicum.

The RNA polymerase subunit RPB10 displays a high level of conservation across archaea and eukarya and is required for cell viability in yeast. Structure determination of this RNA polymerase subunit from Methanobacterium thermoautotrophicum reveals a topology, which we term a zinc-bundle, consisting of three alpha-helices stabilized by a zinc ion. The metal ion is bound within an atypical CX(2)CX(n)CC sequence motif and serves to bridge an N-terminal loop with helix 3. This represents an example of two adjacent zinc-binding Cys residues within an alpha-helix conformation. Conserved surface features of RPB10 include discrete regions of neutral, acidic, and basic residues, the latter being located around the zinc-binding site. One or more of these regions may contribute to the role of this subunit as a scaffold protein within the polymerase holoenzyme.

Amino Acid Sequence↗
PubMed2000

Architecture of RNA polymerase II and implications for the transcription mechanism.

A backbone model of a 10-subunit yeast RNA polymerase II has been derived from x-ray diffraction data extending to 3 angstroms resolution. All 10 subunits exhibit a high degree of identity with the corresponding human proteins, and 9 of the 10 subunits are conserved among the three eukaryotic RNA polymerases I, II, and III. Notable features of the model include a pair of jaws, formed by subunits Rpb1, Rpb5, and Rpb9, that appear to grip DNA downstream of the active center. A clamp on the DNA nearer the active center, formed by Rpb1, Rpb2, and Rpb6, may be locked in the closed position by RNA, accounting for the great stability of transcribing complexes. A pore in the protein complex beneath the active center may allow entry of substrates for polymerization and exit of the transcript during proofreading and passage through pause sites in the DNA.

Amino Acid Motifs↗
PubMed2000

Elongin from Saccharomyces cerevisiae.

Elongin is a transcription elongation factor that was first identified in mammalian systems and is composed of the three subunits, elongin A, B, and C. Sequence homologues of elongin A and elongin C, but not elongin B, were identified in the yeast genome. Neither yeast elongin A nor C sequence homologues was required for cell viability. The two gene products could be purified from yeast as a complex. A recombinant form of the complex, which could only be produced in bacteria if the gene products were co-expressed, was purified over several chromatographic steps. The complex did not stimulate transcription elongation by yeast RNA polymerase II. Using limited proteolysis, the N-terminal 144 residues of yeast elongin A were shown to be sufficient for interaction with yeast elongin C. The purified complex of yeast elongin C/elongin A(1-143) was analyzed using circular dichroism and nuclear magnetic spectroscopy. These studies revealed that yeast elongin A is unfolded but undergoes a dramatic modification of its structure in the presence of elongin C, and that elongin C forms a stable dimer in the absence of elongin A.

Amino Acid Sequence↗
PubMed2000

Yeast RNA polymerase II subunit RPB9. Mapping of domains required for transcription elongation.

The RPB9 subunit of RNA polymerase II regulates transcription elongation activity and is required for the action of the transcription elongation factor, TFIIS. RPB9 comprises two zinc ribbon domains joined by a conserved linker region. The C-terminal zinc ribbon is similar in sequence to that found in TFIIS. To elucidate the relationship between the structure and transcription elongation function of RPB9, we initiated a mutagenesis study on the Saccharomyces cerevisiae homologue. The individual zinc ribbon domains, in isolation or in combination, could not stimulate transcription by a polymerase lacking RPB9, pol IIDelta9. Mutations in the N-terminal zinc ribbon had little effect on transcription activity. By contrast, mutations in the acidic loop that connects the second and third beta-strands of the C-terminal zinc ribbon were completely inactive for transcription. Interestingly, the analogous residues in TFIIS are also critical for elongation activity. A conserved charged stretch in the linker region (residues 89-95, DPTLPR) mediated the interaction with RNA polymerase II.

Amino Acid Sequence↗
PubMed2000

Heme structure and orientation in single monolayers of cytochrome c on polar and nonpolar soft surfaces.

Polarized x-ray absorption fine structure (XAFS) spectroscopy has been performed in fluorescence mode under total external reflection conditions on frozen hydrated single monolayers of yeast cytochrome c (YCC). The protein molecules were vectorially oriented within the monolayer by tethering their naturally occurring and unique surface cysteine residues to the sulfhydryl-endgroups at the surface of a mixed organic self-assembled monolayer, itself covalently attached to an ultrapure silicon wafer. The sulfhydryl-endgroups were isolated by dilution with either methyl- or hydroxyl-endgroups, producing macroscopically nonpolar or uncharged-polar soft surfaces, respectively. Independent information on the heme-plane orientation relative to the monolayer plane was obtained experimentally via optical linear dichroism. The polarized XAFS data have been analyzed both qualitatively and by a global mapping approach limited to systematically altering the various iron-ligand distances within a model for the local atomic environment of the heme prosthetic group, and comparing the theoretically generated XAFS spectra with those obtained experimentally. A similar analysis of unpolarized XAFS data from a frozen solution of YCC was performed using either the heme environment from the NMR solution or the x-ray crystallographic data for YCC as the model structure. All resulting iron-ligand distances were then used in molecular dynamics (MD) computer simulations of YCC in these three systems to investigate the possible effects of anisotropic ligand motions on the fits of the calculated to the experimental XAFS spectra.

Absorptiometry, Photon↗
PubMed2000

Reactivity of monoclonal antibodies against a tryptophan-riboflavin adduct toward irradiated and non-irradiated bovine-eye-lens protein fractions: an indicator of in vivo visible-light-mediated phototransformations.

We describe here the reactivity toward the soluble protein of bovine eye lens of anti-tryptophan-riboflavin (anti-Trp-RF) adduct monoclonal antibodies, which recognize the hapten tryptophan-riboflavin generated by irradiation of a solution of bovine serum albumin in the presence of riboflavin. It is demonstrated that five different anti-Trp-RF adduct monoclonal antibodies, all belonging to the IgG1 isotype, react with the total soluble proteins of bovine eye lens. The components of the soluble protein are separated by Sephadex G-200 chromatography and the isolated fractions analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). All the separated protein fractions also react by a direct ELISA with the monoclonal antibodies; this reaction is more intense when the isolated fractions have been previously irradiated with visible light in the presence of riboflavin under an atmosphere of oxygen or nitrogen. Irradiation of the total soluble protein with visible light in the presence of riboflavin produces the appearance of new bands, corresponding to compounds of higher molecular weight. Riboflavin-sensitized irradiation of the protein fractions with visible light under an oxygen or nitrogen atmosphere is accompanied by a concomitant decrease of the tryptophan fluorescence. It is postulated that the action of visible light in the presence of either the endogenous riboflavin or its derivatives could be partly responsible for the protein aggregation observed during aging.

Animals↗