Abstract
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.
Highlights
TFIIE, TFIIF, and TFIIH) that are thought to function in transcription at all promoters, and the cofactors that mediate transcriptional activation and appear to be more promoterand/or regulatory protein-specific
In previous studies we found that synthesis of a 15-nt RNA from preinitiation complexes limited both the rate and the extent of a single round of transcription by RNA polymerase II at the adenovirus major late promoter (AdMLP) [10]
Given the importance of the early steps of RNA synthesis in limiting the rate and level of transcription, we decided to study the mechanism of early transcription in greater detail using a minimal RNA polymerase II transcription system consisting of TATA binding protein (TBP), TFIIB, TFIIF, RNA polymerase II, and the AdMLP contained on a negatively supercoiled DNA template
Summary
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. TFIIH, in a (d)ATP-dependent reaction, stimulates the fraction of functional complexes that successfully escape the promoter, leading to an increase in the amount of RNA produced [10] These initial studies did not distinguish between initiation and promoter escape nor did they consider other possible steps that may exist during early RNA synthesis. To aid our studies we used competitor DNA that both inhibits an early step in RNA synthesis and limits transcription to a single round This enabled us to characterize a specific transition that occurs after initiation and commits RNA polymerase II to the subsequent step of promoter escape. Because the studies described here were performed in a transcription system containing the minimal number of components necessary to obtain site-specific transcription from the AdMLP (TBP, TFIIB, TFIIF, and core RNA polymerase II), the kinetic model for basal transcription that we established represents the most basic pathway for the RNA polymerase II reaction at this promoter
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