In Vitro Analysis of Promoter Elements Regulating Transcription of the Phosphoenolpyruvate Carboxykinase (GTP) Gene

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 proteinbinding 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.