Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.1.21 (thymidine kinase)
 7,561 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A 42-base pair sequence from the 5' flanking region of the low density lipoprotein receptor gene was shown previously to confer sensitivity to sterol-mediated repression when inserted into the herpes simplex virus thymidine kinase promoter. This sequence contains two contiguous 16-base pair repeats, designated repeats 2 and 3, which differ from each other at four positions. In the current study we have analyzed separately the functions of repeats 2 and 3 by altering their sequences, inserting them into the -60 position of the thymidine kinase promoter, and introducing the hybrid promoters into hamster cells by transfection. These studies show that repeat 3 is a constitutive positive transcriptional element that acts in the absence or presence of sterols. Repeat 2 confers strong repression upon repeat 3 when sterols are present. In vitro DNase footprinting and gel retardation assays show that repeat 3, but not repeat 2, binds purified Sp1, a positive transcription factor. Mutants of repeat 3 that abolish transcriptional activity in vivo abolish Sp1 binding in vitro. We suggest that the low density lipoprotein receptor is regulated by a push-pull mechanism in which sterol-regulated binding of a protein to repeat 2 silences the activity of the adjacent Sp1-binding site in repeat 3.
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PMID:Sterol-dependent repression of low density lipoprotein receptor promoter mediated by 16-base pair sequence adjacent to binding site for transcription factor Sp1. 327 69

Through substitution mutagenesis we identified the promoter elements responsible for basal expression and sterol-mediated repression of transcription of the gene for 3-hydroxy-3-methylglutaryl coenzyme A reductase, a rate-controlling enzyme of cholesterol biosynthesis. Mutant promoters containing 277 base pairs (bp) of reductase 5' flanking sequence were inserted into recombinant plasmids upstream of the coding region for bacterial chloramphenicol acetyltransferase. The plasmids were transfected into hamster fibroblasts, and transcription was measured in the presence and absence of sterols. Mutations in three regions that are known to bind nuclear proteins markedly reduced transcription. Mutation of another protein-binding region of 20 bp in length did not reduce transcription, but it did abolish sterol-mediated repression, producing an operator constitutive phenotype. This mutation also abolished protein binding to the corresponding 20-bp region of DNA as determined by footprinting assays. When a DNA fragment containing these 20 bp was inserted into the herpes simplex virus thymidine kinase promoter, sterol-mediated repression was observed. This sequence contains an octanucleotide that shows a 7/8-bp match with a previously identified regulatory sequence in repeat 2 of the low density lipoprotein receptor promoter, another sterol-repressible gene. We hypothesize that this octanucleotide, GTGGCGGTG, is the core binding site for a sterol-dependent protein that represses transcription.
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PMID:Operator constitutive mutation of 3-hydroxy-3-methylglutaryl coenzyme A reductase promoter abolishes protein binding to sterol regulatory element. 334 49

Sterol regulatory element binding proteins (SREBP-1 and SREBP-2) are attached to the endoplasmic reticulum (ER) and nuclear envelope by a hairpin domain consisting of two transmembrane regions connected by a short lumenal loop of approximately 30 hydrophilic amino acids. In sterol-depleted cells, a protease cleaves the protein in the region of the first transmembrane domain, releasing an NH2-terminal fragment of approximately 500 amino acids that activates transcription of genes encoding the low density lipoprotein receptor and enzymes of cholesterol synthesis. In sterol-overloaded cells, proteolysis does not occur, and transcription is repressed. Through mutational analysis in transfected cells, we identify two segments of SREBPs that are required for proteolysis, one on either side of the ER membrane. An arginine in the lumenal loop is essential. A tetrapeptide sequence (DRSR) on the cytosolic face adjacent to the first transmembrane domain is also required for maximal cleavage. Both of these elements are conserved in the human and hamster versions of SREBP-1 and SREBP-2. Sterol-mediated suppression of cleavage of SREBP-1 was found to be dependent on the extreme COOH-terminal region (residue 1034 to the COOH terminus), which exists in two forms as a result of alternative splicing. The form encoded by the "a" class exons (exons 18a and 19a) undergoes sterol-regulated cleavage. The form encoded by the "c" class exons (18c and 19c) is cleaved less efficiently and is not suppressed by sterols. These studies were made possible through use of a vector that achieves low level expression of epitope-tagged SREBPs under control of the relatively weak thymidine kinase promoter from herpes simplex virus. In contrast to SREBPs overproduced by high level expression vectors, the SREBPs produced at low levels were subject to the same regulated cleavage pattern as the endogenous SREBPs. These results indicate that sterol-regulated proteolysis of SREBPs is a complex process, requiring sequences on both sides of the ER membrane.
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PMID:Regulated cleavage of sterol regulatory element binding proteins requires sequences on both sides of the endoplasmic reticulum membrane. 862 10