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Date of Award

5-2011

Document Type

Campus Access

Degree Name

Doctor of Philosophy (PhD)

Degree Program

Molecular and Cellular Biology

First Advisor

Craig T. Martin

Second Advisor

Robert M. Weis

Third Advisor

Richard W. Vachet

Subject Categories

Biochemistry | Biophysics | Molecular Biology

Abstract

Transcription initiates when the enzyme binds to the promoter region and melts open an initial transcribing bubble that extends from position -4 to +4. The initiation phase continues until the enzyme synthesizes ∼8mer RNA, at which point T7 RNA polymerase begins its transition into the elongation phase. The initiation phase is characterized by a energetic instability, which leads to release of small RNA 2-8 bases in length, known as abortive cycling.

Abortive cycling, which is the release of small RNA transcripts during synthesis of the first 8 bases of a transcript, has been well documented in most single and multisubunit RNA polymerases, and has been shown to occur in vivo. Structural studies have prompted the 'scrunched intermediate' mechanistic model (an elaboration of the earlier stressed intermediate model), which proposes that compaction of the upstream template DNA within the enzyme and/or expansion of the bubble during initiation leads to instability and the release of abortive RNAs. T7 polymerases represent one of the most well characterized transcription systems and despite having no structural similarities to other multi subunit polymerases, shares very similar fundamental mechanistic features. In the initially transcribing abortive phase, the bubble expands as the initial RNA:DNA hybrid grows and the hybrid pushes on components of the enzyme: both key features in the proposed scrunching mechanism. In this work, we directly test predictions of the scrunching model. The introduction of nicks or gaps into the template (scrunched) strand should reduce stress and therefore reduce abortive. Similarly, the introduction of extra bases in this region should increase the release of abortive RNAs or shift their profile to shorter lengths. For all of these modifications, our results show no systematic change in the abortive amounts or profile. An alternate model predicts that a critical source of stress during abortive initiation is the stress caused by the steric clash of the DNA-RNA hybrid against the N-terminal domain. It is already known that this steric clash leads to the transition of the enzyme into a stable elongation complex, however it is unclear as to whether it induces energetic instability within the system. By introducing bulk at the 5' end of an initiating RNA primer we have increased the putative stress of the DNA-RNA primer against the N terminal domain and demonstrated a slight increase in the release of abortive products. The increase in abortive RNA was however not systematic and even with an increase in the steric push, the enzyme continues to transition into the elongation phase and make run off RNA. Our results suggest that abortive cycling is a kinetically controlled process as opposed to any structurally mediated mechanism.

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