Biomedical subjects

T Ellenberger

Publications and source records attributed to T Ellenberger.

29 records · Page 2Linked to original sources

PubMed1998

Crystal structure of a bacteriophage T7 DNA replication complex at 2.2 A resolution.

DNA polymerases change their specificity for nucleotide substrates with each catalytic cycle, while achieving error frequencies in the range of 10(-5) to 10(-6). Here we present a 2.2 A crystal structure of the replicative DNA polymerase from bacteriophage T7 complexed with a primer-template and a nucleoside triphosphate in the polymerase active site. The structure illustrates how nucleotides are selected in a template-directed manner, and provides a structural basis for a metal-assisted mechanism of phosphoryl transfer by a large group of related polymerases.

Bacteriophage T7↗

PubMed1998

Similarities and differences among 105 members of the Int family of site-specific recombinases.

Alignments of 105 site-specific recombinases belonging to the Int family of proteins identified extended areas of similarity and three types of structural differences. In addition to the previously recognized conservation of the tetrad R-H-R-Y, located in boxes I and II, several newly identified sequence patches include charged amino acids that are highly conserved and a specific pattern of buried residues contributing to the overall protein fold. With some notable exceptions, unconserved regions correspond to loops in the crystal structures of the catalytic domains of lambda Int (Int c170) and HP1 Int (HPC) and of the recombinases XerD and Cre. Two structured regions also harbor some pronounced differences. The first comprises beta-sheets 4 and 5, alpha-helix D and the adjacent loop connecting it to alpha-helix E: two Ints of phages infecting thermophilic bacteria are missing this region altogether; the crystal structures of HPC, XerD and Cre reveal a lack of beta-sheets 4 and 5; Cre displays two additional beta-sheets following alpha-helix D; five recombinases carry large insertions. The second involves the catalytic tyrosine and is seen in a comparison of the four crystal structures. The yeast recombinases can theoretically be fitted to the Int fold, but the overall differences, involving changes in spacing as well as in motif structure, are more substantial than seen in most other proteins. The phenotypes of mutations compiled from several proteins are correlated with the available structural information and structure-function relationships are discussed. In addition, a few prokaryotic and eukaryotic enzymes with partial homology with the Int family of recombinases may be distantly related, either through divergent or convergent evolution. These include a restriction enzyme and a subgroup of eukaryotic RNA helicases (D-E-A-D proteins).

Amino Acid Sequence↗

PubMed1998

The mechanism of action of T7 DNA polymerase.

The recent crystal structure determination of T7 DNA polymerase complexed to a deoxynucleoside triphosphate and primer-template DNA has provided the first glimpse of a replicative DNA polymerase in a catalytic complex. The structure complements many functional and structural studies of this and other DNA polymerases, allowing a detailed evaluation of proposals for the mechanism of nucleotidyl transfer and the exploration of the basis for the high fidelity of template-directed DNA synthesis.

Binding Sites↗

PubMed1997

Flexibility in DNA recombination: structure of the lambda integrase catalytic core.

Lambda integrase is archetypic of site-specific recombinases that catalyze intermolecular DNA rearrangements without energetic input. DNA cleavage, strand exchange, and religation steps are linked by a covalent phosphotyrosine intermediate in which Tyr342 is attached to the 3'-phosphate of the DNA cut site. The 1.9 angstrom crystal structure of the integrase catalytic domain reveals a protein fold that is conserved in organisms ranging from archaebacteria to yeast and that suggests a model for interaction with target DNA. The attacking Tyr342 nucleophile is located on a flexible loop about 20 angstroms from a basic groove that contains all the other catalytically essential residues. This bipartite active site can account for several apparently paradoxical features of integrase family recombinases, including the capacity for both cis and trans cleavage of DNA.

Amino Acid Sequence↗

PubMed1997

A good turn for DNA: the structure of integration host factor bound to DNA.

The crystal structure of integration host factor (IHF) complexed with DNA shows how a small heterodimeric protein can induce a big bend in DNA. IHF exerts leverage in the minor groove and wraps DNA around the body of the protein, providing another example of sequence-specific recognition of the minor groove.

Amino Acid Sequence↗

PubMed1995

Pocketing the difference: structures of uracil excision repair proteins.

The recognition of abnormal DNA structure by proteins is fundamentally different from sequence-specific DNA binding. Recent crystal structures of uracil-DNA glycosylases show a novel uracil-binding pocket that explains the enzyme's selectivity for uracil-containing DNA and provide a structural basis for exploring the catalytic mechanism of amino-glycosylic bond cleavage.

DNA↗

PubMed1995

Dual DNA binding specificity of ADD1/SREBP1 controlled by a single amino acid in the basic helix-loop-helix domain.

Adipocyte determination- and differentiation-dependent factor 1 (ADD1), a member of the basic helix-loop-helix (bHLH) family of transcription factors, has been associated with both adipocyte differentiation and cholesterol homeostasis (in which case it has been termed SREBP1). Using PCR-amplified binding analysis, we demonstrate that ADD1/SREBP1 has dual DNA sequence specificity, binding to both an E-box motif (ATCACGTGA) and a non-E-box sequence previously shown to be important in cholesterol metabolism, sterol regulatory element 1 (SRE-1; ATCACCCCAC). The ADD1/SREBP1 consensus E-box site is similar to a regulatory sequence designated the carbohydrate response element, defined by its ability to regulate transcription in response to carbohydrate in genes involved in fatty acid and triglyceride metabolism in liver and fat. When expressed in fibroblasts, ADD1/SREBP1 activates transcription through both the carbohydrate response E-box element and SRE-1. Substitution of an atypical tyrosine in the basic region of ADD1/SREBP1 to an arginine found in most bHLH protein causes a restriction to only E-box binding. Conversely, substitution of a tyrosine for the equivalent arginine in another bHLH protein, upstream stimulatory factor, allows this factor to acquire a dual binding specificity similar to that of ADD1/SREBP1. Promoter activation by ADD1/SREBP1 through the carbohydrate response element E box is not sensitive to the tyrosine-to-arginine mutation, while activation through SRE-1 is completely suppressed. These data illustrate that ADD1/SREBP1 has dual DNA sequence specificity controlled by a single amino acid residue; this dual specificity may provide a novel mechanism to coordinate different pathways of lipid metabolism.

3T3 Cells↗

PubMed1994

Crystal structure of transcription factor E47: E-box recognition by a basic region helix-loop-helix dimer.

A large group of transcription factors regulating cell growth and differentiation share a dimeric alpha-helical DNA-binding domain termed the basic region helix-loop-helix (bHLH). bHLH proteins associate as homodimers and heterodimers having distinctive DNA-binding activities and transcriptional activities that are central to the regulated differentiation of a number of tissues. Some of the bHLH residues specifying these activities have been identified, but a full understanding of their function has awaited further structural information. We report here the crystal structure of the transcription factor E47 bHLH domain bound to DNA. The bHLH of E47 is a parallel, four-helix bundle with structural features that distinguish it from the bHLH-zipper protein Max. The E47 dimer makes nonequivalent contacts to each half of the -CACCTG- binding site. Sequence discrimination at the center of the E box may result from interaction with both the DNA bases and the phosphodiester backbone.

Amino Acid Sequence↗

PubMed1993

Adaptability at the protein-DNA interface is an important aspect of sequence recognition by bZIP proteins.

The related AP-1 and ATF/CREB families of transcriptional regulatory proteins bind as dimers to overlapping or adjacent DNA half-sites by using a bZIP structural motif. Using genetic selections, we isolated derivatives of yeast GCN4 that affect DNA-binding specificity at particular positions of the AP-1 target sequence. In general, altered DNA-binding specificity results from the substitution of larger hydrophobic amino acids for GCN4 residues that contact base pairs. However, in several cases, DNA binding by the mutant proteins cannot be simply explained in terms of the GCN4-AP-1 structure; movement of the protein and/or DNA structural changes are required to accommodate the amino acid substitutions. The quintet of GCN4 residues that make base-pair contacts do not entirely determine DNA-binding specificity because these residues are highly conserved in the bZIP family, yet many of the bZIP proteins bind to distinct DNA sites. The alpha-helical fork between the GCN4 DNA-binding and dimerization surfaces is important for half-site spacing preferences, because mutations in the fork alter the relative affinity for AP-1 and ATF/CREB sites. The basic region in the protein-DNA complex is a long isolated alpha-helix, with no constraints from other parts of a folded domain. From all of these considerations, we suggest that small shifts in position and orientation or local deformations in the alpha-helical backbone distinguish one bZIP complex from another.

Arginine↗

PubMed1990

Folding transition in the DNA-binding domain of GCN4 on specific binding to DNA.

Protein-DNA recognition is often mediated by a small domain containing a recognizable structural motif, such as the helix-turn-helix or the zinc-finger. These motifs are compact structures that dock against the DNA double helix. Another DNA recognition motif, found in a highly conserved family of eukaryotic transcription factors including C/EPB, Fos, Jun and CREB, consists of a coiled-coil dimerization element the leucine-zipper and an adjoining basic region which mediates DNA binding. Here we describe circular dichroism and 1H-NMR spectroscopic studies of another family member, the yeast transcriptional activator GCN4. The 58-residue DNA-binding domain of GCN4, GCN4-p, exhibits a concentration-dependent alpha-helical transition, in accord with previous studies of the dimerization properties of an isolated leucine-zipper peptide. The GCN4-p dimer is approximately 70% helical at 25 degrees C, implying that the basic region adjacent to the leucine zipper is largely unstructured in the absence of DNA. Strikingly, addition of DNA containing a GCN4 binding site (AP-1 site) increases the alpha-helix content of GNC4-p to at least 95%. Thus, the basic region acquires substantial alpha-helical structure when it binds to DNA. A similar folding transition is observed on GCN4-p binding to the related ATF/CREB site, which contains an additional central base pair. The accommodation of DNA target sites of different lengths clearly requires some flexibility in the GCN4 binding domain, despite its high alpha-helix content. Our results indicate that the GCN4 basic region is significantly unfolded at 25 degrees C and that its folded, alpha-helical conformation is stabilized by binding to DNA.

Activating Transcription Factors↗

PubMed1985

Neurotoxic changes in cat neurohypophysis after single and multiple exposures to diisopropylfluorophosphate and soman.

Fine structural changes in cat neurohypophysis have been studied after single and multiple parenteral sub-LD50 doses of the organophosphates diisopropylfluorophosphate (DFP) and pinacolyl methylphosphonofluoridate (soman). While a single sub-LD50 dose of DFP does not alter fine structure, multiple exposures result in degeneration of axons. Single as well as multiple sub-LD50 doses of soman cause dilation of axons with an accumulation of neurosecretory granulated vesicles (NGVs). Increased metabolic activity is suggested by the presence of well-developed Golgi complexes and increased amounts of endoplasmic reticulum in the pituicytes of treated animals. Direct and indirect actions of DFP and soman on neurohypophysial hormone release are discussed as putative mechanisms responsible for the morphological changes in the neurohypophysis.

Animals↗