PubAg

Main content area

In vitro reconstitution of yeast RNA polymerase II transcription initiation with high efficiency

Author:
Fujiwara, Rina, Murakami, Kenji
Source:
Methods 2019
ISSN:
1046-2023
Subject:
DNA, DNA-directed RNA polymerase, immunoblotting, polyacrylamide gel electrophoresis, radiolabeling, staining, transcription factors, transcription initiation, yeasts
Abstract:
Transcription initiation can be reconstituted from highly purified general transcription factors (GTFs), RNA polymerase II (pol II), and promoter DNA. However, earlier biochemical reconstitution systems had a serious technical limitation, namely very poor initiation efficiency. Due to the poor efficiency of the reaction and trace amounts of proteins involved in the pre-initiation complex (PIC) assembly, detection of transcription and PIC formation was only possible by the synthesis of a radiolabeled transcript and by immunoblotting for PIC components on templates. Here we describe a transcription system that is capable of initiating transcription with >90% efficiency of template usage using homogeneous, active yeast components including TFIIA, TFIIB, TBP, TFIIE, TFIIF, TFIIH, Sub1, and pol II. The abundant specifically assembled PICs on promoter DNA can be separated from free general transcription factors (GTFs) and pol II by density gradient sedimentation, irrespective of the length of promoter DNA. The system is robust, and can be modified to accommodate many other transcription factors, and the resulting complexes can be analyzed by SDS-PAGE followed by Coomassie Blue staining. This technical advance now paves the way to conduct definitive biochemical and structural studies of the complete process of pol II initiation from the PIC, through promoter escape, and finally to productive elongation.
Agid:
6352272