Single-molecule studies of prokaryotic and eukaryotic transcription initiation [electronic resource]

Transcription is considered the first step in gene expression. During the transcription initiation phase, a cell decides where and when transcription will occur on its template DNA using a complex network of regulation. Understanding the initiation process is therefore fundamental to any broader understanding of gene regulation. Despite being an active research area for past few decades, fundamental questions regarding mechanistic aspects of initiation remain unanswered. Here, we present findings related to the transcription initiation processes in prokaryotes and eukaryotes, based on single-molecule, real-time observations of RNA polymerase (RNAP) activity using optical tweezers. The only protein complex required to initiate transcription in bacteria is the RNAP holoenzyme itself, which is composed of RNAP core enzyme and a σ factor. The RNAP holoenzyme is able to make multiple contacts with promoter DNA sequences. These contacts modulate both the frequency of transcription, and duration of subsequent steps in the transcription initiation process. Here, we describe a novel single-molecule optical trapping assay that enables us to map the contacts of the Escherichia coli (E. coli) RNAP holoenzyme on the promoter DNA. Using this assay, we were able to observe the initiation process in real time. Through the study, my colleague and I were able to identify strong contacts between the RNAP holoenzyme and promoter DNA at various promoter sequence elements. By monitoring the initiation process from holoenzyme binding to promoter escape, we were also able to observe the remodeling of key contacts between the RNAP holoenzyme and promoter DNA. In contrast to bacterial transcription initiation, the eukaryotic transcription initiation is less studied. A major difference between prokaryotic and eukaryotic transcription initiation is that more proteins are essential to initiate transcription in eukaryotes. To form a minimal, transcription-competent pre-initiation complex (PIC), five general transcription factors (GTFs) are required, in addition to a 12-subunit RNAPII (pol II). Previous biochemical and structural studies have shown that the initiation of pol II transcription proceeds in the following stages: 1) assembly of pol II with GTFs and promoter DNA in a "closed" complex, within which the DNA still remains fully annealed; 2) unwinding about 15 bp of the promoter DNA to form an "open" complex; 3) scanning the downstream DNA sequence for a transcription start site; 4) synthesis of a short RNA transcript, believed to be about 10 nucleotides; 5) and promoter escape. In my thesis, I present findings based on real-time observations of initiation using a reconstituted 32-protein, 1.5 megadalton PIC derived from Saccharomyces cerevisiae. Measurements were made on detailed aspects of the motions of transcription factor IIH (TFIIH), as well as those of pol II, throughout the initiation process. In addition to characterizing the physical properties of TFIIH, including velocity and processivity, our findings establish TFIIH as the motor responsible for start-site scanning. Contrary to expectations, scanning driven by TFIIH entailed the rapid opening of a large transcription bubble, averaging 85 bp, accompanied by the synthesis of a transcript up to the entire length of the extended transcription bubble, followed by promoter escape.

Keywords: Chemistry.
Advisor: Block Steven M.
Cui Bianxiao advisor. Thesis advisor
Moerner W. E. (William Esco)