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Date of Award

9-2009

Document Type

Campus Access

Degree Name

Doctor of Philosophy (PhD)

Degree Program

Chemistry

First Advisor

Craig T. Martin

Second Advisor

Lynmarie K. Thompson

Third Advisor

Michael J. Knapp

Subject Categories

Biochemistry

Abstract

Single subunit T7 RNA polymerase is relatively small and shares mechanistic features with all RNA polymerases, making it an ideal model to study transcription. The T7 RNA polymerase recognizes and binds specifically to a unique sequence in the DNA, melts the DNA at the start site and initiates de novo dinucleotide synthesis. In vitro selection for full function of the T7 RNA polymerase promoter has yielded the consensus sequence, indicating that the latter is optimized for the overall process. However, given potentially competing individual requirements between distinct stages during transcription, the consensus sequence might not be the most tightly binding sequence. In particular, some of the binding energy may be sacrificed to drive unfavorable DNA melting and function may pose additional sequence constraints. I have used SELEX to identify the DNA sequences most tightly binding to T7 RNA polymerase and the results reveal that they are AT-rich in the initial bubble region from positions -4 to +3. Introduction of mismatches in this region lowers the barrier to initial bubble melting, leading to higher binding affinities. That the consensus promoter is G-rich downstream of and including +1 likely reflects the functional need to stabilize the open conformation.

The conformational rearrangement of polymerase facilitates passage from an initially unstable stage to a stable, processive elongation phase now free of the promoter. Indeed, if halted within a homopolymeric template sequence, slippage of the RNA to allow subsequent extension competes favorably with dissociation. This RNA extension is strongly affected by the complex stability, in that removal of a driving force for forward movement of the complex along the DNA promotes slippage, while facilitation of forward movement reduces slippage. The halted elongation complex continues extending RNA by slippage until the substrates are consumed entirely. After depletion of the slippage substrate, the homopolymeric RNA can slide along the template towards its 3' end, forming an arrested elongation complex. Most importantly, this work demonstrates that complex stability arises less from the thermodynamic stability of the hybrid duplex and more from the kinetic stability afforded by the topological locking of the RNA around the template strand DNA.

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