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 hspTO 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 I! 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.[Key Words: C-myc gene; promoter-proximal pausing; RNA polymerase II; transcription initiation; elongation; enhancer]Received November 30, 1994; revised version accepted January 26, 1995.The level of c-myc RNA is regulated largely through a mechanism that operates after transcription initiation. This conclusion is based on the observation that steadystate c-myc RNA levels decline significantly as cells differentiate, whereas transcriptional initiation, as measured in nuclear run-on assays, remains unchanged (Bentley and Groudine 1986; Eick and Bomkamm 1986; Nepveu and Marcu 1986; for review, see Krumm et al. 1993).Recent in vivo assays have demonstrated paused RNA polymerase II complexes at promoter-proximal regions of the c-myc gene. For example, in vivo footprinting with KMnO 4 as a reagent to detect single-stranded DNA residues within transcription bubbles revealed hyper-reactire sites around position +30. These results suggested that RNA polymerases pause in the promoter-proximal region around +30. Nuclear run-on experiments with short probes confirmed that the majority of RNA polymerases is located within the first 169 bp . In another study, nuclear run-on experiments with oligonucleotide probes as short as 50 bp also indi3Corresponding author.cated that the highest level of RNA polymerase II is within the promoter-proximal region between + 1 and +50 (Strobl and Eick 1992). Further experiments demonstrated that sequences 5' of +47 were sufficient to c...