Most known archaeal DNA polymerases belong to the type B family, which also includes the DNA replication polymerases of eukaryotes, but maintain high fidelity at extreme conditions. We describe here the 2.5 Å resolution crystal structure of a DNA polymerase from the Archaea Thermococcus gorgonarius and identify structural features of the fold and the active site that are likely responsible for its thermostable function. Comparison with the mesophilic B type DNA polymerase gp43 of the bacteriophage RB69 highlights thermophilic adaptations, which include the presence of two disulfide bonds and an enhanced electrostatic complementarity at the DNA-protein interface. In contrast to gp43, several loops in the exonuclease and thumb domains are more closely packed; this apparently blocks primer binding to the exonuclease active site. A physiological role of this ''closed'' conformation is unknown but may represent a polymerase mode, in contrast to an editing mode with an open exonuclease site. This archaeal B DNA polymerase structure provides a starting point for structure-based design of polymerases or ligands with applications in biotechnology and the development of antiviral or anticancer agents.Propagation of cells requires faithful DNA replication. This is performed in vivo by DNA polymerases (pols), which attach the appropriate dNTP to the nascent DNA primer strand to match its paired template. Different families of pols are involved in different DNA polymerization processes including not only DNA replication (1, 2) but also repair and recombination (3, 4), a heterogeneity also reflected by varying polypeptide structures and͞or subunit compositions (3, 5). Some pols complement polymerase activity with 3Ј 3 5Ј exonuclease activity (editing activity) and͞or 5Ј 3 3Ј ''structure-specific endonuclease'' activity, often located in separate structural domains on the same polypeptide chain (4-8).Crystal structures are available for most known polymerase families, including the A family DNA polymerases (9-14), pol  (15-17), HIV reverse transcriptase (18)(19)(20), and recently, the B family pol gp43 from bacteriophage RB69 (21). All share a functional polymerase structure, which resembles a right hand built by the palm, fingers and thumb domains (see ref. 7 for review). Although the fingers and thumb domains are highly diverse among the different families, the palm domains, which contain the conserved catalytic aspartate residues, show a similar topology among all families except pol . The polymerase nucleotidyl transfer was studied in detail for the A family polymerases, HIV reverse transcriptase, and pol , and was shown to involve two metal ions (summarized in ref. 7).Considerably less is known for the family of type B pols, which are replicative enzymes in eukaryotes and most likely also Archaea (22,23). The structure of gp43 from bacteriophage RB69 (21) provided an excellent first insight into this family. In addition to the three polymerase domains, gp43 contains an 3Ј 3 5Ј exonuclease domain and an N-termin...
Modulation of gene expression by steroid hormones is mediated by receptor proteins that associate with regulatory elements of responsive genes upon binding the hormone ligand. The finding that two glucocorticoid responsive elements act cooperatively to stimulate transcription of the tyrosine aminotransferase gene prompted us to explore whether synergistic effects also occur when two different steroid hormone receptors are involved. A region of the chicken vitellogenin II gene that displays homologies to glucocorticoid and estradiol responsive elements was tested for its capability to confer estradiol and glucocorticoid inducibility to a heterologous promoter. When positioned immediately upstream of the thymidine kinase gene promoter, this element enhances expression by either steroid. Combination of both hormones results in a synergistic increase of transcription. Mutational analysis shows that sequences that show similarities of glucocorticoid and estradiol responsive elements are absolutely required for hormone induction. Analysis of the dose dependence of induction by both steroids demonstrates that halfmaximal activity is observed at lower hormone concentrations when the other steroid is present in saturating amounts, which suggests that the synergistic induction observed with the combination of hormones is based on a functional interaction of the two hormone receptors.Control of gene expression involves specific interactions of transcription factors with regulatory DNA sequences. Although the mechanisms of activation of eukaryotic genes are poorly understood, it is assumed that cooperative action of transcription factors may be involved as has been demonstrated with prokaryotic systems (1, 2). Activation of transcription by steroid hormones is mediated by receptor proteins that bind to specific sequences of inducible genes after binding of the hormone (3-6). Transcription of the tyrosine aminotransferase gene is controlled by two glucocorticoid responsive elements (GRE) in a cooperative manner (5). This finding prompted us to explore whether synergistic effects also occur when two different steroid hormone receptors are involved.The region between positions -627 and -586 of the chicken vitellogenin II gene contains binding sites for the glucocorticoid and estradiol receptors in close proximity (6, 7). The glucocorticoid receptor binding site contains a sequence that has 10 of 12 base pairs homologous to the consensus proposed for glucocorticoid responsive genes (5, 6). A 15-base-pair oligonucleotide derived from this consensus sequence has been shown to be sufficient for glucocorticoid responsiveness (8). A candidate for an estrogen responsive element (ERE) was first pointed out by sequence comparisons of the vitellogenin genes of frog and chicken (9). This sequence is contained in fragments of the frog vitellogenin A2 and B1 genes and in oligonucleotides that render the thymidine kinase (TK) promoter estrogen-inducible (10-14).We analyzed whether the sequence element of the chicken vitellogenin II ...
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