Poster abstracts

Poster number 36 submitted by Pakoyo F Kamba

RNA binding by the 26.9 kDa Trypanosoma brucei pentatricopeptide repeat protein: insights from deletion mutagenesis and in vitro selection.

Pakoyo F. Kamba (Department of Biochemistry and Molecular Biology, Michigan State University), Neil A. White (Department of Biochemistry and Molecular Biology, Michigan State University), David A. Dickson (Department of Biochemistry and Molecular Biology, Michigan State University), Charles G. Hoogstraten (Department of Biochemistry and Molecular Biology, Michigan State University)

Abstract:
Disease caused by Trypanosoma brucei (T. brucei), the pathogen underlying African Sleeping Sickness, is fatal to humans if untreated. Nevertheless, available drugs are either very toxic or have suboptimal efficacy, meaning novel therapies are needed. Accomplishing this requires understanding the structure and function of essential gene products of the parasite that are absent in the human host. One group of such targets is the sequence-specific RNA binding pentatricopeptide repeat (PPR) family, typified by tandem repeats of 35 amino acids. Most non-plant eukaryotes contain 2 to 6 PPR proteins. T. brucei, however, contains at least 36 [1], a number highly conserved across the trypanosomatids. Unfortunately, utilization of PPR proteins in drug design is hampered by the limited understanding of their structure and mechanism of RNA binding, owing in part to the difficulty in heterologous expression and low aqueous solubility of PPR proteins. Using the smallest T. brucei PPR protein, denoted KRIPP11 [1], we have investigated its RNA ligand by in vitro selection (SELEX). We have probed the sequence and chemical selectivity, minimum size of RNA, and PPR motif requirement for high affinity interaction by gel shift and fluorescence anisotropy assays. As a result, we have an enriched understanding of the KRIPP11-RNA interaction. In addition we have some progress in the crystallization and multidimensional nuclear magnetic resonance (NMR) spectroscopy of KRIPP1.

References:
1. Aphasizheva, I. et al., Mol Cell, 2011. 42(1): p. 106-17.

Keywords: T brucei, PPR, KRIPP11