Date of Award


Degree Type


Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Biochemistry & Molecular Biophysics

First Advisor

Kenji K. Murakami


Transcription by RNA polymerase II (Pol II) is a complex process that requires timely and coordinated regulation at multiple steps for proper gene expression. Initiation is the first step in transcription and decades of biochemical and genome-wide studies have identified proteins involved in the process and revealed their functions. Additionally, technological advancements in cryo-EM enabled researchers to visualize initiation complexes and provide mechanistic insights into initiation processes in the last several years. However, the mechanistic understanding of the transition from transcription initiation to elongation has been limited in part due to the lack of an efficient transcription initiation system in vitro. We purified yeast general transcription factors (GTFs: TFIIA, TFIIB, TBP(a component of TFIID), TFIIE, TFIIF, and TFIIH) and Pol II, all of which are necessary and sufficient for basal transcription initiation, and optimized the initiation system. Using this system, we biochemically re-examined effects of two elongation factors (Cet1-Ceg1 and Spt4/5) on promoter escape, a process in which Pol II dissociates from GTFs except TFIIF for elongation. We find that inclusion of these elongation factors has positive effects on promoter escape. Furthermore, we took advantage of our efficient system, and generated and isolated post-initiation complexes in vitro for structural characterization by cryo-EM. Our structure of the initially-transcribing complex (ITC) stalled +26 shows a large conformational change of TFIIH in the way that it is much closer to TFIIE than in the pre-initiation complex (PIC) and it loses contacts with Pol II. These changes most likely prime for Pol II to escape the promoter. In addition, the structural studies of post-initiation complex stalled +49 reveal two elongation complexes (ECs) colliding to each other as well as show the presence of EC+ITC. In the structure the colliding ECs, the trailing EC contained RNA of ~25 nt in length but has backtracked by ~10 nt upon colliding. These studies together provide a model of the process of promoter escape, where TFIIH can get kicked out by the preceding promoter-proximal EC.

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