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Bachelor of Science, 2001
The Richard Stockton College of New Jersey
Thesis Advisor: William McAllister, Ph.D.
Cell and Molecular Biology Program
Science Center, Room 290
Tuesday, April 27, 2010
Transcription serves as a major control point for gene expression and the different phases of the transcription cycle -- initiation, elongation and termination -- are all important targets for regulation. In this work we sought to characterize the process of transcription by T7 RNA polymerase (T7 RNAP) as it moves through the different phases of the cycle.
The transition from the initiation phase (which requires sequence-specific recognition of the promoter) to the elongation phase (which occurs in a context independent manner) requires major changes in the organization of the transcription complex. To accommodate these different requirements, single subunit RNAPs undergo major structural refolding during promoter release, while multisubunit RNAPs leave behind the subunit responsible for specific initiation. There are two models for refolding of T7 RNAP during this transition. In the first, refolding occurs gradually as the nascent transcript is extended from 2 to 8 nt. In the second, most of the changes are delayed until promoter release, after 8 nt of RNA have been synthesized.
To examine the refolding pathway, we mutated regions of the enzyme that are altered during the transition and examined the effects of these changes on transcription initiation. One series of mutations focused upon a region that would affect the movement of a “core” subdomain (which rotates as a rigid body during the transition), and another targeted a region that interacts with the template strand of the DNA in the EC but not in the IC. Both series of mutations resulted in enhanced release of abortive products at 5-7 nt, supporting a model in which the refolding process occurs in a stepwise manor, with some changes occurring prior to promoter release.
Formally, the pathway to termination can be thought of as a reversal of the process that occurs when RNAP moves from an initiation competent form to an elongation competent form. T7 RNA polymerase (RNAP) recognizes two classes of pause/termination signals: structure dependent (class I) signals in which the nascent RNA forms a stem loop structure (similar to intrinsic signals utilized by bacterial RNAPs), and sequence specific (class II) signals in which there is no apparent secondary structure in the RNA.
In this work we examined sequence-specific pausing by T7 RNAP at a class II signal found in the concatamer junction (CJ) of replicating T7 DNA. In vivo, the CJ signal induces a pause by RNAP, which is greatly extended in the presence of T7 lysozyme. The paused complex is thought to recruit other proteins that are required for processing and packaging of the DNA into infections phage particles. CJ-like signals have been identified in DNA molecules that are not related to T7 (e.g., the human preparathyroid hormone, PTH, gene as well as in the rrnB T1 intrinsic termination signal in E. coli). However, these signals result in efficient termination, not pausing, and are physiologically unrelated to phage development.
We demonstrate here that the addition of T7 lysozyme to RNAP paused at the CJ signal results in an extremely stable complex that remains associated with the template and RNA for up to 60 min. Biochemical assays such as KMnO4 and Exo III footprinting indicate that the halted complexes share similar attributes to an elongation complex (EC). In addition, we examined the effects of topology on signal recognition and found that the sequence in the nontemplate (NT) strand contributes more to signal recognition than the sequence in the template (T) strand, and that certain bases in the signal are particularly important for pausing and/or response to lysozyme. These findings are discussed in the context of various models of pausing or termination that have been proposed for this type of signal.