Promoter-proximal pausing of RNA polymerase II defines a general rate-limiting step after transcription initiation.

We have shown previously that the majority of RNA polymerase II complexes initiated at the c-myc gene are paused in the promoter-proximal region, similar to observations in the Drosophila hsp70 gene. Our analyses define the TATA box or initiator sequences in the c-myc gene as necessary components for the establishment of paused RNA polymerase II. Deletion of upstream sequences or even the TATA box does not influence significantly the degree of transcriptional initiation or pausing. Deletion of both the TATA box and sequences at the transcription initiation site, however, abolishes transcriptional pausing of transcription complexes but still allows synthesis of full-length RNA. Further analyses with synthetic promoter constructs reveal that the simple combination of upstream activator with TATA consensus sequences or initiator sequences act synergistically to recruit high levels of RNA polymerase II complexes. Only a minor fraction of these complexes escapes into regions further downstream. Several different trans-activation domains fused to GAL4-DNA-binding domains, including strong activators such as VP16, do not eliminate promoter-proximal pausing of RNA polymerase. Thus, we conclude that pausing of RNA polymerase II is a common phenomenon in eukaryotic transcription and does not require complex promoter structures. Further analyses reveal that enhancers have a modest influence on transcription initiation and on release of transcription complexes out of the pause site but may function primarily to increase the elongation competence of transcription complexes.