A kinetic model for the early steps of RNA synthesis by human RNA polymerase II

Eukaryotic mRNA synthesis is a highly regulated process involving numerous proteins acting in concert with RNA polymerase II to set levels of transcription from individual promoters. The transcription reaction consists of multiple steps beginning with preinitiation complex formation and ending in the production of a full-length primary transcript. We used pre-steady-state approaches to study the steps of human mRNA transcription at the adenovirus major late promoter in a minimal in vitro transcription system. These kinetic studies revealed an early transition in RNA polymerase II transcription, termed escape commitment, that occurs after initiation and prior to promoter escape. Escape commitment is rapid and is characterized by sensitivity to competitor DNA. Upon completion of escape commitment, ternary complexes are resistant to challenge by competitor DNA and slowly proceed forward through promoter escape. Escape commitment is stimulated by transcription factors TFIIE and TFIIH. We measured forward and reverse rate constants for discrete steps in transcription and present a kinetic model for the mechanism of RNA polymerase II transcription that describes five distinct steps (preinitiation complex formation, initiation, escape commitment, promoter escape, and transcript elongation) and clearly shows promoter escape is rate-limiting in this system.