Promoter clearance by RNA polymerase II is an extended, multistep process strongly affected by sequence

We have characterized RNA polymerase II complexes halted from +16 to +49 on two templates which differ in the initial 20 nucleotides (nt) of the transcribed region. On a template with a purine-rich initial transcript, most complexes halted between +20 and +32 become arrested and cannot resume RNA synthesis without the SII elongation factor. These arrested complexes all translocate upstream to the same location, such that about 12 to 13 bases of RNA remain in each of the complexes after SII-mediated transcript cleavage. Much less arrest is observed over this same region with a second template in which the initially transcribed region is pyrimidine rich, but those complexes which do arrest on the second template also translocate upstream to the same location observed with the first template. Complexes stalled at +16 to +18 on either template do not become arrested. Complexes stalled at several locations downstream of +35 become partially arrested, but these more promoter-distal arrested complexes translocate upstream by less than 10 nt; that is, they do not translocate to a common, far-upstream location. Kinetic studies with nonlimiting levels of nucleoside triphosphates reveal strong pausing between +20 and +30 on both templates. These results indicate that promoter clearance by RNA polymerase II is at least a two-step process: a preclearance escape phase extending up to about +18 followed by an unstable clearance phase which extends over the formation of 9 to 17 more bonds. Polymerases halted during the clearance phase translocate upstream to the preclearance location and arrest in at least one sequence context.