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Jillian A. Parker
December 3, 2010
Major: Biochemistry
Project Sponsor: Dr. Thomas C. Leeper
Number of Credits: 2
Site-directed Mutagenesis of the Protein-Protein Contacts in the Yeast mRNA Processing
Complex
The complex yeast genome requires highly specific protein complexes to direct and
ensure correct transcription product formation. Hrp1 and Rna15 are two complex stabilizing
proteins which have been shown to interact in the yeast cleavage and polyadenylation complex
during correct mRNA product formation. Without these two RNA binding proteins, incorrect
mRNA synthesis, and consequently incorrect protein products, may result. This research
project attempts to characterize the interaction of Hrp1, Rna15, and their shared RNA to
discover the exact mechanism of correct mRNA formation in yeast. Specifically, a mutation in
either protein should disrupt ternary complex formation and lead to incorrect 3’-end mRNA
processing. The D193N Hrp1 mutant and 15N isotope labeled wild-type Rna15 were expressed
in Escherichia coli and purified for study via nuclear magnetic resonance (NMR) spectroscopy.
Both one dimensional (1D) and two dimensional (2D) NMR experiments were conducted to
characterize the interaction between Hrp1 and Rna15 and to monitor their RNA binding
capabilities. Interestingly, this specific Hrp1 D193N mutant did not disrupt complex formation
between it and Rna15 and still allowed this ternary protein complex to bind to its correct RNA
recognition sequence. Overall, this project successfully demonstrates the protein-protein
interactions in the yeast mRNA processing complex.