Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The ALCR protein is the transcriptional activator of the ethanol utilization pathway in the filamentous fungus Aspergillus nidulans. This activator belongs to a family of fungal proteins having a conserved DNA-binding domain containing six cysteines (C6 class) with some striking features. At variance with other motifs of this class, the binding domain of ALCR is strongly asymmetrical in relation to the central cysteines and moreover was predicted to adopt a helix-turn-helix structure. This domain of ALCR was synthesized in Escherichia coli and purified as a glutathione-S-transferase fusion protein. Our results show that the transcriptional activator ALCR is a DNA-binding protein. The DNA-binding motif contains zinc that is necessary for the specific DNA binding. The ALCR peptide binds upstream of the coding region of alcR to two specific targets with different affinities that are characterized by a conserved 5-nucleotide core, 5'-CCGCA-3' (or its reverse). One site, the lower-affinity binding site, is a direct repeat, and the other, the higher-affinity binding site, is a palindromic sequence with dyad symmetry. Therefore, the ALCR binding protein is able to recognize one DNA sequence in two different configurations. An alcR mutant obtained by deletion of the two specific targets in the cis-acting region of the alcR gene is unable to grow on ethanol and does not express any alcohol dehydrogenase activity. These results demonstrate that the binding sites are in vivo functional targets (UASalc) for the ALCR protein in A. nidulans. They corroborate prior evidence that alcR is autoregulated.
Mol Cell Biol 1992 May
PMID:Identification of the promoter region involved in the autoregulation of the transcriptional activator ALCR in Aspergillus nidulans. 156 30

The eukaryotic transcriptional repressor PRDI-BF1 contains five zinc fingers of the C2H2 type, and the protein binds specifically to PRDI, a 14-bp regulatory element of the beta interferon gene promoter. We have investigated the amino acid sequence requirements for specific binding to PRDI and found that the five zinc fingers and a short stretch of amino acids N terminal to the first finger are necessary and sufficient for PRDI-specific binding. The contribution of individual zinc fingers to DNA binding was investigated by inserting them in various combinations into another zinc finger-containing DNA-binding protein whose own fingers had been removed. We found that insertion of PRDI-BF1 zinc fingers 1 and 2 confer PRDI-binding activity on the recipient protein. In contrast, the insertion of PRDI-BF1 zinc fingers 2 through 5, the insertion of zinc finger 1 or 2 alone, and the insertion of zinc fingers 1 and 2 in reverse order did not confer PRDI-binding activity. We conclude that the first two PRDI-BF1 zinc fingers together are sufficient for the sequence-specific recognition of PRDI.
Mol Cell Biol 1992 May
PMID:Only two of the five zinc fingers of the eukaryotic transcriptional repressor PRDI-BF1 are required for sequence-specific DNA binding. 156 31

We showed previously that the increased rate of mRNA turnover associated with premature translational termination in the yeast Saccharomyces cerevisiae requires a functional UPF1 gene product. In this study, we show that the UPF1 gene codes for a 109-kDa primary translation product whose function is not essential for growth. The protein contains a potential zinc-dependent nucleic acid-binding domain and a nucleoside triphosphate-binding domain. A 300-amino-acid segment of the UPF1 protein is 36% identical to a segment of the yeast SEN1 protein, which is required for endonucleolytic processing of intron-containing pre-tRNAs. The same region is 32% identical to a segment of Mov-10, a mouse protein of unknown function. Dominant-negative upf1 mutations were isolated following in vitro mutagenesis of a plasmid containing the UPF1 gene. They mapped exclusively at conserved positions within the sequence element common to all three proteins, whereas the recessive upf1-2 mutation maps outside this region. The clustering of dominant-negative mutations suggests the presence of a functional domain in UPF1 that may be shared by all three proteins. We also identified upf mutations in three other genes designated UPF2, UPF3, and UPF4. When alleles of each gene were screened for effects on mRNA accumulation, we found that the recessive mutation upf3-1 causes increased accumulation of mRNA containing a premature stop codon. When mRNA half-lives were measured, we found that excess mRNA accumulation was due to mRNA stabilization. On the basis of these results, we suggest that the products of at least two genes, UPF1 and UPF3, are responsible for the accelerated rate of mRNA decay associated with premature translational termination.
Mol Cell Biol 1992 May
PMID:Gene products that promote mRNA turnover in Saccharomyces cerevisiae. 156 46

DNA binding domain proteins (DBDP) were prepared using a pET construct containing an insert coding for amino acids 49-122 of human thyroid hormone receptor (hTR) alpha and 103-179 of hTR beta. These proteins were expressed in Escherichia coli strain BL21 (DE3)-plysS after induction by isopropyl-D-thiogalactopyranoside (IPTG). The hTR alpha and hTR beta DBDP contain respectively 79 and 82 amino acids, including an amino terminal 4 amino acid extension derived from pET-3a or the synthesized initiation codon. Using a gel shift assay, both DBDPs were found to bind to a DNA oligonucleotide containing a thyroid hormone response element (TRE). The DBDPs competed with full length hTR alpha 1 for binding to the oligonucleotide. Apo-DBDPs (Zn2+ released by low pH) failed to bind to the palindromic TRE. DNA binding is restored however if apo-DBDP is preincubated in 500 microM Zn2+. When the DBDPs were expressed in COS-7 cells using a pCB6+ expression vector, they did not induce expression of a TRE-CAT fusion gene. hTR DBDPs thus can bind to DNA, presumably as monomers, since they do not contain the leucine zipper-like motif for dimerization. In COS-7 cells, they fail to cause transactivation of a TRE-CAT fusion gene. It is inferred that this may be because the DBDPs are not translocated to the nucleus or lack a transactivation domain.
Mol Cell Endocrinol 1992 Apr
PMID:Expression and function of a human thyroid hormone receptor-derived DNA-binding domain protein. 158 92

STP1 is an unessential yeast gene involved in the removal of intervening sequences from some, but not all, families of intervening sequence-containing pre-tRNAs. Previously, we proposed that STP1 might encode a product that generates pre-tRNA conformations efficiently recognized by tRNA-splicing endonuclease. To test the predictions of this model, we have undertaken a molecular analysis of the STP1 gene and its products. The STP1 locus is located on chromosome IV close to at least two other genes involved in RNA splicing: PRP3 and SPP41. The STP1 open reading frame (ORF) could encode a peptide of 64,827 Da; however, inspection of putative transcriptional and translational regulatory signals and mapping of the 5' ends of mRNA provide evidence that translation of the STP1 ORF usually initiates at a second AUG to generate a protein of 58,081 Da. The STP1 ORF contains three putative zinc fingers. The first of these closely resembles both the DNA transcription factor consensus and the Xenopus laevis p43 RNA-binding protein consensus. The third motif more closely resembles the fingers found in spliceosomal proteins. Employing antisera to the endogenous STP1 protein and to STP1-LacZ fusion proteins, we show that the STP1 protein is localized to nuclei. The presence of zinc finger motifs and the nuclear location of the STP1 protein support the model that this gene product is involved directly in pre-tRNA splicing.
Mol Cell Biol 1992 Jun
PMID:STP1, a gene involved in pre-tRNA processing, encodes a nuclear protein containing zinc finger motifs. 158 61

ADR1 is a yeast transcription factor that contains two zinc fingers of the Cys-2-His-2 (C2H2) class. Mutations that change the specificity of DNA binding of ADR1 to its target site, upstream activation sequence 1 (UAS1), have been identified at three positions in the first zinc finger. Mutations Arg-115 to Gln, His-118 to Thr, and Arg-121 to Asn led to new specificities of DNA binding at adjacent positions 10, 9, and 8 (3'-GAG-5') in UAS1. Arg-115 is at the finger tip, and His-118 and Arg-121 are at positions 3 and 6, respectively, in the alpha helix of finger 1. One double mutant displayed the binding specificity expected from the properties of its constituent new-specificity mutations. Mutations in the second finger that allowed its binding site to be identified through loss-of-contact phenotypes were made. These mutations imply a tail-to-tail orientation of the two ADR1 monomers on their adjacent binding sites. Finger 1 is aligned on UAS1 in an amino-to-carboxyl-terminal orientation along the guanine-rich strand in a 3'-to-5' direction. One of the ADR1 mutants was functional in vivo with both its cognate binding site and wild-type UAS1, but the other two mutants were defective in transactivation despite their ability to bind with high affinity to their cognate binding sites.
Mol Cell Biol 1992 Jun
PMID:Mutations in the zinc fingers of ADR1 that change the specificity of DNA binding and transactivation. 158 70

Sequences of 47 members of the Zn-containing alcohol dehydrogenase (ADH) family were aligned progressively, and an evolutionary tree with detailed branch order and branch lengths was produced. The alignment shows that only 9 amino acid residues (of 374 in the horse liver ADH sequence) are conserved in this family; these include eight Gly and one Val with structural roles. Three residues that bind the catalytic Zn and modulate its electrostatic environment are conserved in 45 members. Asp 223, which determines specificity for NAD, is found in all but the two NADP-dependent enzymes, which have Gly or Ala. Ser or Thr 48, which makes a hydrogen bond to the substrate, is present in 46 members. The four Cys ligands for the structural zinc are conserved except in zeta-crystallin, the sorbitol dehydrogenases, and two bacterial enzymes. Analysis of the evolutionary tree gives estimates of the times of divergence for different animal ADHs. The human class II (pi) and class III (chi) ADHs probably diverged about 630 million years ago, and the newly identified human ADH6 appeared about 520 million years ago, implying that these classes of enzymes may exist or have existed in all vertebrates. The human class I ADH isoenzymes (alpha, beta, and gamma) diverged about 80 million years ago, suggesting that these isoenzymes may exist or have existed in all primates. Analysis of branch lengths shows that these plant ADHs are more conserved than the animal ones and that class III ADHs are more conserved than class I ADHs. The rate of acceptance of point mutations (PAM units) shows that selection pressure has existed for ADHs, implying that these enzymes play definite metabolic roles.
J Mol Evol 1992 Jun
PMID:Progressive sequence alignment and molecular evolution of the Zn-containing alcohol dehydrogenase family. 159 44

The structure of Cu,Zn yeast superoxide dismutase has been determined to 2.5 A resolution. The enzyme crystallizes in the P2(1)2(1)2 space group with two dimeric enzyme molecules per asymmetric unit. The structure has been solved by molecular replacement techniques using the dimer of the bovine enzyme as the search model, and refined by molecular dynamics with crystallographic pseudo-energy terms, followed by conventional crystallographic restrained refinement. The R-factor for 32,088 unique reflections in the 10.0 to 2.5 A resolution range (98.2% of all possible reflections) is 0.158 for a model comprising two protein dimers and 516 bound solvent molecules, with a root-mean-square deviation of 0.016 A from the ideal bond lengths, and an average B-factor value of 29.9 A2. A dimeric molecule of the enzyme is composed of two identical subunits related by a non-crystallographic 2-fold axis. Each subunit (153 amino acid residues) has as its structural scaffolding a flattened antiparallel eight-stranded beta-barrel, plus three external loops. The overall three-dimensional structure is quite similar to the phylogenetically distant bovine superoxide dismutase (55% amino acid homology), the largest deviations can be observed in the regions of amino acid insertions. The major insertion site hosting residues Ser25A and Gly25B, occurs in the 2,3 beta-turn between strands 2b and 3c, resulting in the structural perturbations of the two neighbouring strands. The second insertion site, at the end of the 3c beta-strand in the wide Greek-key loop, hosts the Asn35A residue, having an evident effect on the structure of the loop and possibly on the neighbouring 5,4 beta-turn. The salt bridge Arg77-Asp99 and the disulphide bridge Cys55-Cys144 stabilize the loop regions containing the metal ligands. The stereochemistry of the two metal centres is conserved, with respect to the bovine enzyme. The Cu2+ ligands show an uneven distortion from a square plane, while Zn2+ co-ordination geometry is distorted tetrahedral. The imidazole ring of the His61 residue forms a bridge between Cu and Zn ions. A solvent peak compatible with a fifth ligand is observed 2.0 A away from the copper in the active site channel, which is filled by ordered water molecules that possibly contribute to the stability and function of the enzyme. The charged residues responsible for the electrostatic guidance of the substrate to the active site (Glu130, Glu131, Lys134 and Arg141) are fairly conserved in their positions, some of them showing different interactions in the four chains due to the intermolecular contacts between the dimers.(ABSTRACT TRUNCATED AT 400 WORDS)
J Mol Biol 1992 Jun 05
PMID:Crystal structure of yeast Cu,Zn superoxide dismutase. Crystallographic refinement at 2.5 A resolution. 160 82

Zinc fingers are usually associated with proteins that interact with DNA. Yet in two oocyte-specific Xenopus proteins, TFIIA and p43, zinc fingers are used to bind 5S RNA. One of these, TFIIIA, also binds the 5S RNA gene. Both proteins have nine zinc fingers that are nearly identical with respect to size and spacing. We have determined the relative affinities of groups of zinc fingers from TFIIIA for both 5S RNA and the 5S RNA gene. We have also determined the relative affinities of groups of zinc fingers from p43 for 5S RNA. The primary protein regions for RNA and DNA interaction in TFIIIA are located at opposite ends of the molecule. All zinc fingers from TFIIIA participate in binding 5S RNA, but zinc fingers from the C terminus have the highest affinity. N-terminal zinc fingers are essential for binding the 5S RNA gene. In contrast, zinc fingers at the amino terminus of p43 are essential for binding 5S RNA.
Mol Cell Biol 1992 Jul
PMID:Differential binding of zinc fingers from Xenopus TFIIIA and p43 to 5S RNA and the 5S RNA gene. 162 Jan 23

We show that the DNA-binding domain of the Drosophila melanogaster regulatory protein Tramtrack consists of a 66 amino acid sequence containing two zinc-finger motifs and a short sequence N-terminal to the first finger motif. This short N-terminal sequence is essential for DNA binding and we suggest it is involved in maintaining the three-dimensional structure of the first finger domain, as has been seen in the nuclear magnetic resonance structure of one of the zinc-finger domains of the yeast transcription factor SW15. The characterization of the DNA-binding activity of this 66 residue peptide (delta 911zf) shows that it binds in a sequence-specific manner, as a monomer, to a natural target site with an apparent KD approximately 4 x 10(-7) M. The shortest delta 911zf binding site, which retains full affinity, consists of an 11 base-pair sequence with a one nucleotide overhang at each 5' end. DNase I, hydroxyl radical and methylation protection footprinting studies show that, in common with other zinc-finger proteins, delta 911zf binds in the major groove of DNA. The data presented are consistent with the zinc-fingers of Tramtrack contacting both strands of the DNA, and thus the binding differs in detail to that observed in the crystal structure of the three zinc-fingers of Zif268 complexed to their target DNA.
J Mol Biol 1992 Jul 20
PMID:Sequence-specific DNA binding by a two zinc-finger peptide from the Drosophila melanogaster Tramtrack protein. 164 Apr 55


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