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Biomedical subjects

A M Edwards

Publications and source records attributed to A M Edwards.

At least 73 records · Page 4Linked to original sources
PubMed1998

Yeast transcript elongation factor (TFIIS), structure and function. I: NMR structural analysis of the minimal transcriptionally active region.

TFIIS is a general transcription elongation factor that helps arrested RNA polymerase II elongation complexes resume transcription. We have previously shown that yeast TFIIS (yTFIIS) comprises three structural domains (I-III). The three-dimensional structures of domain II and part of domain III have been previously reported, but neither domain can autonomously stimulate transcription elongation. Here we report the NMR structural analysis of residues 131-309 of yTFIIS which retains full activity and contains all of domains II and III. We confirm that the structure of domain II in the context of fully active yTFIIS is the same as that determined previously for a shorter construct. We have determined the structure of the C-terminal zinc ribbon domain of active yTFIIS and shown that it is similar to that reported for a shorter construct of human TFIIS. The region linking domain II with the zinc ribbon of domain III appears to be conformationally flexible and does not adopt a single defined tertiary structure. NMR analysis of inactive mutants of yTFIIS support a role for the linker region in interactions with the transcription elongation complex.

Magnetic Resonance Spectroscopy↗
PubMed1998

Yeast transcript elongation factor (TFIIS), structure and function. II: RNA polymerase binding, transcript cleavage, and read-through.

The transcriptionally active fragment of the yeast RNA polymerase II transcription elongation factor, TFIIS, comprises a three-helix bundle and a zinc ribbon motif joined by a linker region. We have probed the function of this fragment of TFIIS using structure-guided mutagenesis. The helix bundle domain binds RNA polymerase II with the same affinity as does the full-length TFIIS, and this interaction is mediated by a basic patch on the outer face of the third helix. TFIIS mutants that were unable to bind RNA polymerase II were inactive for transcription activity, confirming the central role of polymerase binding in the TFIIS mechanism of action. The linker and zinc ribbon regions play roles in promoting cleavage of the nascent transcript and read-through past the block to elongation. Mutation of three aromatic residues in the zinc ribbon domain (Phe269, Phe296, and Phe308) impaired both transcript cleavage and read-through. Mutations introduced in the linker region between residues 240 and 245 and between 250 and 255 also severely impaired both transcript cleavage and read-through activities. Our analysis suggests that the linker region of TFIIS probably adopts a critical structure in the context of the elongation complex.

Models, Molecular↗
PubMed1998

Repeated tertiary fold of RNA polymerase II and implications for DNA binding.

X-ray diffraction data from two forms of yeast RNA polymerase II crystals indicate that the two largest subunits of the polymerase, Rpb1 and Rpb2, may have similar folds, as is suggested by secondary structure predictions. DNA may bind between the two subunits with its 2-fold axis aligned to a pseudo 2-fold axis of the protein.

Amino Acid Sequence↗
PubMed1998

The RPA32 subunit of human replication protein A contains a single-stranded DNA-binding domain.

Replication protein A (RPA) is a conserved nuclear single-stranded DNA (ssDNA)-binding protein. Human RPA (hRPA) comprises three subunits of approximately 70, 32, and 14 kDa (hRPA70, hRPA32 and hRPA14). RPA is known to bind ssDNA through two ssDNA-binding domains in the RPA70 subunit. Here, we demonstrate that the complex of hRPA32 and hRPA14 has an ssDNA-binding domain. Limited proteolysis of the hRPA14.32 complex defined a core dimer composed of the central region of hRPA32 (amino acids 43-171) and RPA14. The core dimer bound ssDNA with an affinity of approximately 10-50 microM, which is at least 100-fold more avid than the DNA-binding affinity of the intact dimer. Analysis of the predicted secondary structure of hRPA32 suggests that amino acids 63-150 of hRPA32 form an ssDNA-binding domain similar in structure to each of those in hRPA70. The complex of hRPA14 and hRPA32-(43-171) in turn formed a trimeric complex with the C-terminal region of hRPA70 (amino acids 436-616). The ssDNA-binding affinity of this trimeric complex was 3 to 5-fold higher than hRPA14.32-(43-171) alone, suggesting a role for the C terminus of hRPA70 in ssDNA binding.

Amino Acid Sequence↗
PubMed1998

Vectorially oriented monolayers of the cytochrome c/cytochrome oxidase bimolecular complex.

Vectorially oriented monolayers of yeast cytochrome c and its bimolecular complex with bovine heart cytochrome c oxidase have been formed by self-assembly from solution. Both quartz and Ge/Si multilayer substrates were chemical vapor deposited with an amine-terminated alkylsiloxane monolayer that was then reacted with a hetero-bifunctional cross-linking reagent, and the resulting maleimide endgroup surface then provided for covalent interactions with the naturally occurring single surface cysteine 102 of the yeast cytochrome c. The bimolecular complex was formed by further incubating these cytochrome c monolayers in detergent-solubilized cytochrome oxidase. The sequential formation of such monolayers and the vectorially oriented nature of the cytochrome oxidase was studied via meridional x-ray diffraction, which directly provided electron density profiles of the protein(s) along the axis normal to the substrate plane. The nature of these profiles is consistent with previous work performed on vectorially oriented monolayers of either cytochrome c or cytochrome oxidase alone. Furthermore, optical spectroscopy has indicated that the rate of binding of cytochrome oxidase to the cytochrome c monolayer is an order of magnitude faster than the binding of cytochrome oxidase to an amine-terminated surface that was meant to mimic the ring of lysine residues around the heme edge of cytochrome c, which are known to be involved in the binding of this protein to cytochrome oxidase.

Algorithms↗
PubMed1998

The efficacy of slow versus faster inhalation of cromolyn sodium in protecting against allergen challenge in patients with asthma.

BACKGROUND: Approximately one third of patients with allergy-induced asthma who are treated with aerosolized cromolyn sodium (CS) fail to achieve a full therapeutic effect. This lack of effectiveness could involve nonhomogeneous distribution of drug in the lung as a result of high inspiratory flow rates. OBJECTIVE: We sought to determine the efficacy of slow versus faster inhalation of CS in protecting against allergen challenge in patients with asthma. METHODS: Eight patients with asthma underwent two allergen challenges 30 minutes after pretreatment with CS that was inhaled from a large holding chamber at approximately 30 L/min or approximately 70 L/min. Percent decreases in FEV1 at a common dose of allergen on the two challenge days were compared. Values of skew (an indicator of aerosol distribution homogeneity) obtained from gamma camera lung images after slow and faster inhalation of radiolabeled CS were also compared. RESULTS: Mean (+/- SD) allergen-induced decrease in FEV1 was 5.4% +/- 4.2% after slow inspiration of CS, which was significantly less than the allergen-induced decrease in FEV1 after faster inhalation of CS with 12.6% +/- 11% (p < 0.05). Mean skew values were also significantly decreased after slow inspiration of CS, and differences in decreases in allergen FEV1 and skew values for the two breathing maneuvers were significantly correlated. CONCLUSION: These data indicate that protection against allergen-induced asthma can be optimized by slowly inspiring CS from a large holding chamber compared with faster inhalation of the drug. These results appear to be related to enhanced distribution homogeneity of CS within the lungs.

Administration, Inhalation↗
PubMed1998

Origin DNA-binding proteins.

The first step in DNA replication involves the recognition of origin DNA sequences by origin-binding proteins. The three-dimensional structures of three different origin DNA-binding proteins have recently been solved. These proteins form a structural class distinct from other DNA-binding proteins. One of the origin-binding proteins, Epstein-Barr virus nuclear antigen 1, most likely has two modes of DNA binding; the sequential use of these modes may be important for the initiation of DNA replication.

Antigens, Polyomavirus Transforming↗
PubMed1998

Gleb von Anrep.

Explore the source record for details and available documents.

Asthma↗
PubMed1997

Transcription elongation through DNA arrest sites. A multistep process involving both RNA polymerase II subunit RPB9 and TFIIS.

The role of yeast RNA polymerase II (pol II) subunit RPB9 in transcript elongation was investigated by examining the biochemical properties of pol II lacking RPB9 (pol IIDelta9). The maximal rate of chain elongation was nearly identical for pol II and pol IIDelta9. By contrast, pol IIDelta9 elongated more efficiently through DNA sequences that signal the elongation complex to pause or arrest. The addition of purified recombinant RPB9 to pol IIDelta9 restored the elongation properties of the mutant polymerase to those of the wild-type enzyme. Arrested pol IIDelta9 complexes were refractory to levels of TFIIS that promoted maximal read-through with pol II. However, both pol II and pol IIDelta9 complexes stimulated with TFIIS undergo transcript cleavage, confirming that transcript cleavage and read-through activities can be uncoupled. Our observations suggest that both TFIIS and RPB9 are required to stimulate the release of RNA polymerase II from the arrested state.

Cloning, Molecular↗
PubMed1997

Structure of the single-stranded-DNA-binding domain of replication protein A bound to DNA.

The single-stranded-DNA-binding proteins (SSBs) are essential for DNA function in prokaryotic and eukaryotic cells, mitochondria, phages and viruses. The structures of four SSBs have been solved, but the molecular details of the interaction of SSBs with DNA remain speculative. We report here the crystal structure at 2.4 A resolution of the single-stranded-DNA-binding domain of human replication protein A (RPA) bound to DNA. Replication protein A is a heterotrimeric SSB that is highly conserved in eukaryotes. The largest subunit, RPA70, binds to single-stranded (ss)DNA and mediates interactions with many cellular and viral proteins. The DNA-binding domain, which lies in the middle of RPA70, comprises two structurally homologous subdomains oriented in tandem. The ssDNA lies in a channel that extends from one subdomain to the other. The structure of each RPA70 subdomain is similar to those of the bacteriophage SSBs, indicating that the mechanism of ssDNA-binding is conserved.

Amino Acid Sequence↗
PubMed1997

Replication protein A. Characterization and crystallization of the DNA binding domain.

Replication protein A (RPA) is a heterotrimeric single-stranded DNA-binding protein in eukaryotic cells. The DNA binding activity of human RPA has been previously localized to the N-terminal 441 amino acids of the 70-kDa subunit, RPA70. We have used a combination of limited proteolysis and mutational analysis to define the smallest soluble fragment of human RPA70 that retains complete DNA binding activity. This fragment comprises residues 181-422. RPA181-422 bound DNA with the same affinity as the 1-441 fragment and had a DNA binding site of 8 nucleotides or less. RPA70 fragments were subjected to crystal trials in the presence of single-stranded DNA, and diffraction quality crystals were obtained for RPA181-422 bound to octadeoxycytidine. The RPA181-422 co-crystals belonged to the P2(1)2(1)2(1) space group, with unit cell dimensions of a = 34.3 A, b = 78.0 A, and c = 95.4 A and diffracted to a resolution of 2.1 A.

Crystallography, X-Ray↗
PubMed1996

In vitro characterization of mutant yeast RNA polymerase II with reduced binding for elongation factor TFIIS.

We have reported previously the isolation and genetic characterization of mutations in the gene encoding the largest subunit of yeast RNA polymerase II (RNAPII), which lead to 6-azauracil (6AU)-sensitive growth. It was suggested that these mutations affect the functional interaction between RNAPII and transcription-elongation factor TFIIS because the 6AU-sensitive phenotype of the mutant strains was similar to that of a strain defective in the production of TFIIS and can be suppressed by increasing the dosage of the yeast TFIIS-encoding gene, PPR2, RNAPIIs were purified and characterized from two independent 6AU-sensitive yeast mutants and from wild-type (wt) cells. In vitro, in the absence of TFIIS, the purified wt polymerase and the two mutant polymerases showed similar specific activity in polymerization, readthrough at intrinsic transcriptional arrest sites and nascent RNA cleavage. In contrast to the wt polymerase, both mutant polymerases were not stimulated by the addition of a 3-fold molar excess of TFIIS in assays of promoter-independent transcription, readthrough or cleavage. However, stimulation of the ability of the mutant RNAPIIs to cleave nascent RNA and to read through intrinsic arrest sites was observed at TFIIS:RNAPII molar ratios greater than 600:1. Consistent with these findings, the binding affinity of the mutant polymerases for TFIIS was found to be reduced by more than 50-fold compared with that of the wt enzyme. These studies demonstrate that TFIIS has an important role in the regulation of transcription by yeast RNAPII and identify a possible binding site for TFIIS on RNAPII.

Binding Sites↗
PubMed1996

Elongation factor TFIIS contains three structural domains: solution structure of domain II.

Transcription elongation by RNA polymerase II is regulated by the general elongation factor TFIIS. This factor stimulates RNA polymerase II to transcribe through regions of DNA that promote the formation of stalled ternary complexes. Limited proteolytic digestion showed that yeast TFIIS is composed of three structural domains, termed I, II, and III. The two C-terminal domains (II and III) are required for transcription activity. The structure of domain III has been solved previously by using NMR spectroscopy. Here, we report the NMR-derived structure of domain II: a three-helix bundle built around a hydrophobic core composed largely of three tyrosines protruding from one face of the C-terminal helix. The arrangement of known inactivating mutations of TFIIS suggests that two surfaces of domain II are critical for transcription activity.

Fungal Proteins↗
PubMed1996

Crystal structure of the DNA-binding domain of the Epstein-Barr virus origin-binding protein, EBNA1, bound to DNA.

The Epstein-Barr virus nuclear antigen 1 (EBNA1) protein binds to and activates DNA replication from oriP, the latent origin of DNA replication in Epstein-Barr virus. The crystal structure of the DNA-binding domain of EBNA1 bound to an 18 bp binding site was solved at 2.4 A resolution. EBNA1 comprises two domains, a flanking and a core domain. The flanking domain, which includes a helix that projects into the major groove and an extended chain that travels along the minor groove, makes all of the sequence-determining contacts with the DNA. The core domain, which is structurally homologous to the complete DNA-binding domain of the bovine papilloma virus E2 protein, makes no direct contacts with the DNA bases. A model for origin unwinding is proposed that incorporates the known biochemical and structural features of the EBNA1-origin interaction.

Amino Acid Sequence↗
PubMed1996

Dystrophic neurite formation associated with age-related beta amyloid deposition in the neocortex: clues to the genesis of neurofibrillary pathology.

The formation of dystrophic neurites associated with beta amyloid plaques in Alzheimer's disease (AD) appears to involve a transformation of normal neuronal cytoskeletal proteins. In order to investigate what may be the earliest neuronal changes associated with the development of dystrophic neurites, we have examined the neurochemical profile of abnormal neuritic processes associated with the beta amyloid deposition in non-AD, aged cases. In all non-AD individuals demonstrating some degree of beta amyloid deposition in the superior frontal gyrus, clustered swollen and ring-like structures, located principally in layers II and III, were labeled with antibodies to phosphorylated and nonphosphorylated domains of the middle and high molecular weight neurofilament subunits. These abnormal neurites were not immunolabeled for tau or ubiquitin or stained with thioflavine S. Double labeling for neurofilaments and thioflavine S confirmed that these clusters of dystrophic neurites were associated with plaque-like deposits. These results show that anatomically and neurochemically specific forms of dystrophic neurites can occur in non-AD cases that contain beta amyloid deposition. If these abnormal neurites correspond to an immature form of the dystrophic neurites found in the neuritic plaques of Alzheimer's disease, then neurofibrillary pathology associated with this disease may begin with an initial misprocessing and accumulation of neurofilament proteins. Furthermore, these data are consistent with the proposal that the development of neurofibrillary pathology may begin with neurofilamentous hypertrophy in damaged distal processes followed by reactive changes in the cell bodies of origin of these fibers involving cytoskeletal alterations that ultimately lead to neurofibrillary tangle formation.

Aged↗