Using a HeLa cell nuclear extract (NE)-based in vitro runoff transcription system, we have examined the effect of Sp1 on the activation of a TATA-containing chimeric DNA polymerase beta (pAS8) promoter. The results demonstrated that the TATA element-dependent basal activity of the pAS8 promoter was stimulated 4-fold by supplementation of a Sp1-depleted HeLa cell nuclear extract (NEd) with purified human Sp1, indicating that pAS8 promoter activity is dependent upon Sp1. A detailed kinetic analysis based on a three-step kinetic model of transcription initiation showed that Sp1 stimulates the activity of the pAS8 promoter by increasing the amount of closed preinitiation complex (RP(c)) assembly as well as by enhancing the rate of promoter clearance (k(3)). There was no significant effect of Sp1 on the apparent rate of open complex (RP(o)) formation (k(2)) of the pAS8 promoter. These studies define more precisely the kinetic mechanisms by which Sp1 may regulate the rate of transcript formation of a TATA-containing promoter.