This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).
Intellectual Merit The ability to modulate specific steps in gene expression via "designer" regulatory elements has broad application in basic research and biotechnology. RNA binding proteins can modulate RNA-mediated events, but most classes of RNA binding protein are poor candidates for engineering applications due to the difficulty of predicting their RNA binding properties from their sequence or structure. In this context, the unusual RNA recognition mechanism of the Pumilio Homology Domain (PUM-HD) has attracted considerable attention: the PUM-HD consists of eight helical repeats that recognize a contiguous 8-9 nucleotide RNA segment via a one-repeat, one-nucleotide mechanism. This modular recognition mechanism suggested a "code" for RNA recognition by the PUM-HD, and offers the possibility of engineering the PUM-HD to bind novel RNA targets. This project focuses on a less well-known protein class, the pentatricopeptide repeat (PPR) proteins, which offer greater promise for the design of novel RNA binding specificities and for the engineering of gene regulatory systems. PPR proteins are predicted to adopt a helical repeat solenoid structure that is reminiscent of the PUM-HD. Current data suggest that PPR tracts, like the PUM-HD, bind RNA via a modular recognition mechanism whose rules should be definable. However, the highly variable number of repeats in PPR proteins and the remarkable diversity of their natural RNA ligands and physiological functions support the view that PPR tracts provide a much more malleable platform for RNA binding. Furthermore, the ability of PPR proteins to bind single-stranded RNA along an unusually long interface imparts an unusual repertoire of biochemical activities, which predict specific effects on gene expression when PPR proteins are targeted to specific sites. This project will (i) test the premise that the unusual properties of PPR proteins can be exploited to modulate gene expression at diverse steps in diverse organisms, and (ii) make substantial progress towards understanding the "code" for sequence-specific RNA binding by PPR proteins.
Broader Impacts Development of the PPR motif as a platform for engineering applications will require the ability to design PPR proteins to recognize a wide variety of RNAs, and knowledge about the steps in gene expression that can be modulated by PPR/RNA interactions. This project addresses both of these issues, and will thereby provide the foundation for the development of new tools for the manipulation of gene expression in both prokaryotes and eukaryotes. Successful outcomes will provide the basis for the design of powerful selection schemes for the directed evolution of PPR proteins to recognize new RNA ligands. Furthermore, the results will provide mechanistic insights into this large and poorly-understood family of RNA binding proteins, which plays numerous essential roles in organellar gene expression (and thus in energy transduction) in all eukaryotes. The interdisciplinary nature of this research and the diversity of the organisms and assays to be employed will provide a rich educational experience for an undergraduate student. The same student will participate throughout the two-year project, such that the student can take ownership of one aim and follow it through from beginning to end.
