Nuclei isolated from H4IIE rat
hepatoma cells were used in an in vitro run-on assay, with probes directed against various regions of the
phosphoenolpyruvate carboxykinase [GTP:
oxaloacetate carboxy-lyase (transphosphorylating); EC 4.1.1.32] gene, to analyze whether transcription proceeds uniformly across this gene in response to
insulin and cAMP treatment. Fewer polymerase II complexes were associated with the
phosphoenolpyruvate carboxykinase gene after
insulin treatment, as compared with cAMP-treated cells, but they were distributed uniformly, so
insulin does not block transcription at a discrete site, nor does it cause gradual, but progressive, premature termination. The
phosphoenolpyruvate carboxykinase primary transcript was synthesized at a rate of about 2500
nucleotides per min in cAMP-treated cells and about 1000
nucleotides per min in
insulin-treated cells. Thus
insulin retards transcript elongation in comparison with cAMP, but this action does not account for the total effect
insulin has on transcription. After
insulin treatment, few, if any, nascent transcripts are associated with the first 69
nucleotides of the gene, whereas in cAMP-treated cells the opposite is true. These observations lead us to suggest that both
insulin and cAMP exert their primary effects directly at the level of transcription initiation, but in opposite ways.