Abstract
Extrinsic transcription termination typically involves remodeling of RNA polymerase by an accessory helicase. In yeast this is accomplished by the Sen1 helicase homologous to human senataxin (SETX). To gain insight into these processes we develop a DNA scaffold construct compatible with magnetic-trapping assays and from which S. cerevisiae RNA polymerase II (Pol II), as well as E. coli RNA polymerase (ecRNAP), can efficiently initiate transcription without transcription factors, elongate, and undergo extrinsic termination. By stalling Pol II TECs on the construct we can monitor Sen1-induced termination in real-time, revealing the formation of an intermediate in which the Pol II transcription bubble appears half-rewound. This intermediate requires ~40 sec to form and lasts ~20 sec prior to final dissociation of the stalled Pol II. The experiments enabled by the scaffold construct permit detailed statistical and kinetic analysis of Pol II interactions with a range of cofactors in a multi-round, high-throughput fashion.
Highlights
Extrinsic transcription termination typically involves remodeling of RNA polymerase by an accessory helicase
Two E. coli his terminators, analogous to the E. coli tR2 terminator previously shown to terminate polymerase II (Pol II) transcription[20], that are positioned flanking the permanent bubble, and oriented in such a way as to capture transcription initiated in either direction from the bubble
The complex is viewed through the formation and dissolution of the topological transcription bubble universally formed by RNA polymerases
Summary
Extrinsic transcription termination typically involves remodeling of RNA polymerase by an accessory helicase. Promoter-dependent Pol II initiation has been sidestepped by pre-assembling Pol II on RNA/DNA scaffolds to form TECs with higher efficiency and study the mechanisms of elongation in high-resolution optical trapping systems[9,12] Because these are complex single-round assays in which each DNA molecule can be transcribed only once, relatively low data collection throughput represents a major limitation of such assays. We present a methodology based on a recyclable scaffold DNA construct[18,19], which permits multiround analysis of transcription, extending the spatial resolution and data throughput made possible by magnetic trap nanomanipulation to the eukaryotic transcription systems We use this scaffold construct to characterize structure–function relations in the Pol II elongation complex and study important aspects of elongation, such as the action of the elongation factor TFIIS and the dynamics of R-loop formation. We employ our system to reveal important features of the transcription termination activity of the conserved helicase Sen[1], a key actor in the control of pervasive transcription
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