An 8 nt RNA triggers a rate-limiting shift of RNA polymerase II complexes into elongation

Abstract

To better understand the critical conversions that RNA polymerase II complexes undergo during promoter escape, we determined in vitro the precise positions of the ratelimiting step and the last step requiring negative superhelicity or TFIIE and TFIIH. We found that both steps occur after synthesis of an 8 nt RNA during the stage encompassing translocation of the polymerase active site to the ninth register. When added to reactions just before this step, TFIIE and TFIIH overcame the requirement for negative superhelicity. The positions at which both steps occur were strictly dependent on RNA length as opposed to the location of the polymerase relative to promoter elements, showing that the transcript itself controls transformations during promoter escape. We propose a model in which completion of promoter escape involves a ratelimiting conversion of early transcribing complexes into elongation complexes. This transformation is triggered by synthesis of an 8 nt RNA, occurs independent of the general transcription factors, and requires under-winding in the DNA template via negative superhelicity or the action of TFIIE and TFIIH.

TATA-binding Protein and Transcription Factor IIB Induce Transcript Slipping during Early Transcription by RNA Polymerase II

“…The promoter DNA influences the level of transcription and the position at which RNA synthesis begins through sequences in the regulatory and core promoter elements (1,2). In addition, the conformation of promoter DNA can regulate the level of transcription, both in the presence and absence of histones (3)(4)(5)(6)(7). The general transcription factors, including TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH, are required for pol II to transcribe in a specific fashion in most, if not all, genes (8).…”

“…Regulated genespecific transcription in cells requires a plethora of additional factors, including activators, repressors, co-regulators, and chromatin-modifying factors (2,6,8,9). In addition to the promoter and protein factors, biochemical studies have shown that the RNA transcript is not simply the product of the reaction, but it also stabilizes elongation complexes through the RNA: DNA hybrid and influences transformations that occur during early transcription (3,10,11). Although the core factors required for pol II to synthesize a transcript in vitro are understood, structural and biochemical studies are still uncovering new information about the mechanism by which this process occurs and is regulated.…”

“…We previously found that the points at which escape commitment and the rate-limiting step occur are set by the length of the transcript as opposed to the position of the pol II active site on the DNA template (3,11). We speculated that this might be a general feature of transitions that occur during early transcription; hence we asked whether the point in the reaction at which transcript slippage occurs is set by the length of the RNA (5 nt) or by the position of the pol II active site on the DNA (register ϩ5).…”

“…Transcription of mRNA by pol 3 II is a complex process that involves the highly regulated interplay of promoter DNA, many protein factors, and the transcript RNA itself. The promoter DNA influences the level of transcription and the position at which RNA synthesis begins through sequences in the regulatory and core promoter elements (1,2).…”

“…Kinetic studies have identified the rate-limiting step in transcription in vitro as occurring during promoter escape (3,24). Experiments monitoring the melted region of DNA during promoter escape have shown that the upstream edge of the transcription bubble initially remains anchored while the downstream edge extends, thereby increasing the size of the bubble (4, 22).…”