Operator constitutive mutation of 3-hydroxy-3-methylglutaryl coenzyme A reductase promoter abolishes protein binding to sterol regulatory element

T. F. Osborne, G. Gil, J. L. Goldstein, M. S. Brown

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125 Citations (Scopus)

Abstract

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, GTG(C)(G)GGTG, is the core binding site for a sterol-dependent protein that represses transcription.

Original languageEnglish (US)
Pages (from-to)3380-3387
Number of pages8
JournalJournal of Biological Chemistry
Volume263
Issue number7
StatePublished - 1988

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Sterol Regulatory Element Binding Proteins
Sterols
Transcription
Oxidoreductases
Base Pairing
Mutation
Protein Binding
Plasmids
Genes
Mutagenesis
Chloramphenicol O-Acetyltransferase
Thymidine Kinase
LDL Receptors
5' Flanking Region
DNA
Biosynthesis
Simplexvirus
Fibroblasts
Nuclear Proteins
Viruses

ASJC Scopus subject areas

  • Biochemistry

Cite this

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title = "Operator constitutive mutation of 3-hydroxy-3-methylglutaryl coenzyme A reductase promoter abolishes protein binding to sterol regulatory element",
abstract = "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, GTG(C)(G)GGTG, is the core binding site for a sterol-dependent protein that represses transcription.",
author = "Osborne, {T. F.} and G. Gil and Goldstein, {J. L.} and Brown, {M. S.}",
year = "1988",
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journal = "Journal of Biological Chemistry",
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T1 - Operator constitutive mutation of 3-hydroxy-3-methylglutaryl coenzyme A reductase promoter abolishes protein binding to sterol regulatory element

AU - Osborne, T. F.

AU - Gil, G.

AU - Goldstein, J. L.

AU - Brown, M. S.

PY - 1988

Y1 - 1988

N2 - 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, GTG(C)(G)GGTG, is the core binding site for a sterol-dependent protein that represses transcription.

AB - 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, GTG(C)(G)GGTG, is the core binding site for a sterol-dependent protein that represses transcription.

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