PROMOTER ESCAPE BY RNA-POLYMERASE-II - FORMATION OF AN ESCAPE-COMPETENT TRANSCRIPTIONAL, INTERMEDIATE IS A PREREQUISITE FOR EXIT OF POLYMERASE FROM THE PROMOTER

Shortly after initiating promoter-specific transcription in vitro, mam malian RNA polymerase II becomes highly susceptible td arrest in a pro moter-proximal region 9-13 base pairs downstream of the transcriptiona l start site (Dvir, A., Conaway, R. C., and Conaway, J. W. (1996) J. B iol, Chem, 271, 23352-23356), Arrest by polymerase in this region is s uppressed by TFIIH in an ATP-dependent reaction (Dvir, A.; Conaway, R. C., and Conaway, J. W. (1997) Proc. Natl. Acad, Sci, U. S. A. 94, 900 6-9010), In this report, we present evidence that, in addition to TFII H and an ATP cofactor, efficient transcription by RNA polymerase II th rough this promoter-proximal region requires formation of an ''escape- competent'' transcriptional intermediate, Formation of this intermedia te requires template DNA 40-50 base pairs downstream of the transcript ional start site. This requirement for downstream DNA is transient, si nce template DNA downstream of +40 is dispensable for assembly of the preinitiation complex, for initiation and synthesis of the first 10-12 phosphodiester bonds of nascent transcripts and for further extension of transcripts longer than similar to 14 nucleotides, Thus, promoter escape requires that the RNA polymerase, II transcription complex unde rgoes a critical structural transition, likely driven by interaction o f one or more components of the transcriptional machinery with templat e DNA 40-50 base pairs downstream of the transcriptional start site.