In vitro analysis of promoter elements regulating transcription of the phosphoenolpyruvate carboxykinase (GTP) gene

Dwight J. Klemm, William J. Roesler, Jinsong Liu, Edwards Park, Richard W. Hanson

Research output: Contribution to journalArticle

22 Citations (Scopus)

Abstract

A cell-free system for the study of transcription from the promoter of the phosphoenolpyruvate carboxykinase (GTP) gene by using nuclear extracts from rat tissues was developed. The level of basal transcription from the phosphoenolpyruvate carboxykinase (PEPCK) promoter between -490 and +73 was highest when extracts from liver nuclei, rather than kidney, spleen, and HeLa nuclear extracts, were used. A series of 5′ deletions and block mutations were also tested for their effects on basal transcription in vitro. The promoter truncated to -355 had the highest rate of basal transcription, while subsequent deletion to -277 markedly decreased the rate of transcription. Further deletion of the promoter to -134 resulted in a twofold increase in the basal level of transcription compared with that of the promoter deleted to -277. However, subsequent deletion of the NF-1-CCAAT-binding transcription factor binding site or the proximal cyclic AMP (cAMP) regulatory element caused a decrease in basal transcription. Block mutations were inserted into nine specific protein-binding regions of the PEPCK promoter previously shown to be of functional significance or to bind nuclear proteins. Mutation of the TATA box resulted in a 94% decrease in the level of transcription noted with the intact promoter, while sequence substitutions within the proximal cAMP regulatory element decreased the transcription rate to 25%. The addition of the catalytic subunit of cAMP-dependent protein kinase to the in vitro system stimulated transcription from the intact promoter or from a promoter deletion to -109. However, a promoter deletion to -68, which removes the proximal cAMP regulatory element, was unresponsive to added protein kinase catalytic subunit. These findings indicate that the PEPCK promoter between -490 and +73 contains sequences responsive to hormonal and tissue-specific factors in nuclei from rat tissues. The sensitivity of this in vitro transcription system closely mimics the processes regulating PEPCK transcription in rat tissues and should make it ideal for testing the function of purified transcription factors.

Original languageEnglish (US)
Pages (from-to)480-485
Number of pages6
JournalMolecular and cellular biology
Volume10
Issue number2
DOIs
StatePublished - Jan 1 1990

Fingerprint

Phosphoenolpyruvate Carboxykinase (GTP)
Phosphoenolpyruvate
Cyclic AMP
Genes
CCAAT-Binding Factor
Cyclic AMP-Dependent Protein Kinase Catalytic Subunits
Liver Extracts
TATA Box
Mutation
Tissue Extracts
Cell-Free System
Sequence Deletion
Thromboplastin
Nuclear Proteins
Protein Binding
Protein Kinases
Catalytic Domain
Transcription Factors
Spleen
Binding Sites

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Cell Biology

Cite this

In vitro analysis of promoter elements regulating transcription of the phosphoenolpyruvate carboxykinase (GTP) gene. / Klemm, Dwight J.; Roesler, William J.; Liu, Jinsong; Park, Edwards; Hanson, Richard W.

In: Molecular and cellular biology, Vol. 10, No. 2, 01.01.1990, p. 480-485.

Research output: Contribution to journalArticle

Klemm, Dwight J. ; Roesler, William J. ; Liu, Jinsong ; Park, Edwards ; Hanson, Richard W. / In vitro analysis of promoter elements regulating transcription of the phosphoenolpyruvate carboxykinase (GTP) gene. In: Molecular and cellular biology. 1990 ; Vol. 10, No. 2. pp. 480-485.
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