Identification of basal and cyclic AMP regulatory elements in the promoter of the phosphoenolpyruvate carboxykinase gene
Promoter elements important for basal and cyclic AMP (cAMP)-regulated expression of the phosphoenolpyruvate carboxykinase (PEPCK) gene have been identified by analysis of a series of PEPCK promoter mutations in transfection experiments. Fusion genes containing wild-type and mutated PEPCK promoter sequences from -600 to +69 base pairs (bp) fused to the coding sequence for chloramphenicol acetyltransferase were studied. Internal deletion mutations that replaced specific bases with a 10-bp linker within the region from - 129 bp to - 18 bp of the PEPCK promoter were examined. In addition, wild-type and mutated DNA templates were used as probes in DNase I protection experiments to determine sites of protein-DNA interaction. The PEPCK promoter contains a binding site for nuclear factor 1-CAAT. Deletion of the 5' end of this binding site reduced the size of the DNase I footprint in this region but had no effect on promoter activity. In contrast, deletion or disruption of the 3' end of this binding site completely eliminated protein binding and reduced promoter activity by 50%. Deletion of core sequences of the cAMP regulatory element (CRE) resulted in loss of cAMP responsiveness and an 85% decrease in basal promoter activity, indicating that the CRE also functions as a basal stimulatory element.
- Research Organization:
- Dept. of Molecular Physiology and Biophysics, Vanderbilt Univ., Nashville, TN (US)
- OSTI ID:
- 7095919
- Journal Information:
- Mol. Cell. Biol.; (United States), Vol. 8:8
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
GENE OPERONS
GENE MUTATIONS
PHOSPHOTRANSFERASES
GENE REGULATION
AMP
CYCLASES
DNA SEQUENCING
DNA-ASE
ENZYME ACTIVITY
EXPERIMENTAL DATA
GENES
MUTATIONS
PHENOTYPE
PYRUVIC ACID
CARBOXYLIC ACIDS
DATA
ENZYMES
ESTERASES
HYDROLASES
INFORMATION
KETO ACIDS
LYASES
NUCLEOTIDES
NUMERICAL DATA
ORGANIC ACIDS
ORGANIC COMPOUNDS
PHOSPHODIESTERASES
PHOSPHORUS-GROUP TRANSFERASES
STRUCTURAL CHEMICAL ANALYSIS
TRANSFERASES
550200* - Biochemistry