FUNCTIONAL AND BIOPHYSICAL CHARACTERIZATION OF TRYPANOSOMA BRUCEI PENTATRICOPEPTIDE REPEAT PROTEINS By Pakoyo Fadhiru KambaShuttling of Trypanosoma brucei between the contrasting environments of the mammalian blood stream and the arthropod vector is aided by a tunable mitochondrion which downregulates its activity in the blood stream and upregulates it in the arthropod gut via differential expression of its genes. Trypanosomes, however, are deficient in transcription factors and regulate gene expression posttranscriptionally via transcript stability, differential processing, and translation. It is hypothesized that a milieu of RNA binding proteins are involved, most of which are still putative. In this thesis, I investigated the function and mode of action of T. brucei pentatricopeptide repeat (PPR) proteins, a family of á-helical, organellar RNA binding proteins characterized by tandem repeats of 35 amino acids. I had three aims: (i) to elucidate the RNA binding specificity of two T. brucei PPR proteins, one with a molecular mass of 27 kDa (PPR27) and another with a molecular mass of 41 kDa (PPR41); (ii) to understand the contribution of each PPR motif to PPR27-RNA binding; (iii) to develop suitable methods for production of concentrated, monodisperse, PPR proteins for structural studies. For all aims, recombinant protein was produced in Escherichia coli and used for in vitro experiments. First, the RNA binding specificity of PPR27 was determined. We found that PPR27 selectively binds single-stranded polyguanosine RNA quadruplexes over other sequences and nucleic acid forms. PPR27 binding did not disrupt the quadruplex structure commonly adopted by poly-G sequences. There is selective enrichment of G-tracts in maxicircle genes for extensively edited mRNAs, suggesting recognition of G-rich RNA was biologically relevant. A pull-down assay identified uncleaved RNA precursors as biological ligands. An association between PPR27 and the small mitoribosomal subunit was recently reported. Hence, PPR27 may aid crosstalk between pre-mRNA processing and translation. Next, the RNA binding activity and solubility of PPR27 deprived of one or more PPR motifs was analyzed. Deletion of PPR motifs modestly reduced the affinity of PPR27 for RNA, and RNA binding still occurred with only two intact PPR motifs. Except for the construct with only two PPR motifs, which suffered a 7-fold drop in RNA binding affinity for G12 ssRNA, the RNA binding Kd of truncated variants ranged between 2.5- and 3.5-fold that of wild type. Thus, all the PPR modules in PPR27 are seemingly involved in RNA contacts. Finally, screening of truncated PPR27 variants identified one soluble enough to attain structural biology concentrations.Thirdly, the RNA binding specificity of PPR41 was studied. Under non-equilibrium conditions, stable binding was only observed with poly(G) ssRNA. However, under equilibrium conditions PPR41 showed strong and similar affinities for G12, U12, and (GGU)4, modest affinity for A12, and reduced affinity for C12 ssRNAs. PPR41 pulled down precursor RNA transcripts from a mitochondrial RNA extract. RNA editing inserts U's into G-tracts. A role for PPR41 in either edited RNA binding or precursor RNA processing is therefore likely. Lastly, I probed the application to PPR proteins of some production systems which have previously been successful with some difficult proteins. I found that wild type PPR27 can be purified and refolded from inclusion bodies of its thioredoxin fusion protein, and showed that chimeras of PPR proteins with large solubility tags have promise for NMR spectroscopic and crystallization studies.
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