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The Role of a Nuclear-Encoded DEAD-box Protein from Saccharomyces cerevisiae in Mitochondrial Group I Intron Splicing

Bifano, Abby Lynn Shumaker

Abstract Details

2010, Doctor of Philosophy, Case Western Reserve University, Biochemistry.

DEAD-box proteins are a large class of ATPases that are involved in almost all aspects of RNA metabolism. Many of these enzymes have been shown to possess common biochemical properties in vitro, including the ability to bind and hydrolyze ATP, to bind nucleic acids, and to unwind and anneal nucleic acids strands and, therefore, DEAD-box proteins are generally thought to modulate RNA structure in vivo. To a large degree, however, the functions of individual DEAD-box proteins in vivo remain unclear and, furthermore, it is unknown which of the aforementioned properties are important for their activities in vivo. Thus, it is critical to define the mechanisms by which DEAD-box proteins function with their native substrates, in order to understand the permissible roles of these important enzymes in RNA metabolism and, more broadly, gene expression.

The work presented here is an investigation into the role of a representative DEAD-box protein, Mss116p, and the mechanisms by which it functions in the splicing of a native group I intron substrate, the aI5β intron from the mitochondrial COX1 gene in S. cerevisiae. In the first study, we show that Mss116p, in an ATP-dependent manner, specifically facilitates the second step of aI5β splicing in vitro and that its role in exon ligation is temporally controlled and dependent on the “upstream” activity of another RNA binding protein, Mrs1p. We also demonstrate that the ability of Mss116p to efficiently hydrolyze ATP, rather than unwind a stable model duplex, is critical for its function in aI5β splicing.

In the second study, we explored the catalytic properties of Mss116p that were required for the splicing of the aI5β intron, as well as additional native intron substrates, in vivo. Similar to our biochemical results, we found that efficient ATPase activity, rather than robust unwinding activity, was required for Mss116p’s role in the splicing of all examined intron substrate in vivo. Given that ATP hydrolysis stimulates the recycling of DEAD-box proteins, our observations support a model in which enzyme turnover is a crucial factor in the splicing function of Mss116p.

Martin Snider, PhD (Advisor)
Jeff Coller, PhD (Committee Chair)
Alan Tartakoff, PhD (Committee Member)
Jonatha Gott, PhD (Committee Member)
Pieter deHaseth, PhD (Committee Member)
177 p.

Recommended Citations

Citations

  • Bifano, A. L. S. (2010). The Role of a Nuclear-Encoded DEAD-box Protein from Saccharomyces cerevisiae in Mitochondrial Group I Intron Splicing [Doctoral dissertation, Case Western Reserve University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=case1283302534

    APA Style (7th edition)

  • Bifano, Abby. The Role of a Nuclear-Encoded DEAD-box Protein from Saccharomyces cerevisiae in Mitochondrial Group I Intron Splicing. 2010. Case Western Reserve University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=case1283302534.

    MLA Style (8th edition)

  • Bifano, Abby. "The Role of a Nuclear-Encoded DEAD-box Protein from Saccharomyces cerevisiae in Mitochondrial Group I Intron Splicing." Doctoral dissertation, Case Western Reserve University, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=case1283302534

    Chicago Manual of Style (17th edition)