Characterization of the promoter of the human G_i2 α-subunit gene

The promoter of the human gene for the α-subunit of G_i2, a GTP-binding signal transduction protein, was analyzed by cloning the 5′ flanking region of the gene upstream of the bacterial chloramphenicol acetyltransferase (CAT) gene and measuring the level of CAT expression after transfection in CV-1 green monkey kidney cells. Analysis of multiple 5′ deletion mutants reveals that a minimum of 85 bases upstream of the major transcriptional initiation site are required for full basal promoter activity. Deleting 11 bases to position -74 causes a 4-fold decrease in promoter activity. Another major decrement in activity is seen when a GC box between -46 and -33 is deleted, consistent with this region being a functionally active Sp1 factor-binding site. Primer extension of a CAT-specific primer hybridized to RNA from cells transfected with a G_i2α promoter-CAT construct confirms approximately the same transcriptional start site as the endogenous G_i2α gene. The 3′ deletion mutants that either approach or delete the transcriptional start site have markedly diminished activity.