Mutational analysis of Trypanosoma brucei editosome proteins KREPB4 and KREPB5 reveals domains critical for function.

The transcriptome of kinetoplastid mitochondria undergoes extensive RNA editing that inserts and deletes uridine residues (U's) to produce mature mRNAs. The editosome is a multiprotein complex that provides endonuclease, TUTase, exonuclease, and ligase activities required for RNA editing. The editosome's KREPB4 and KREPB5 proteins are essential for editosome integrity and parasite viability and contain semi-conserved motifs corresponding to zinc finger, RNase III, and PUF domains, but to date no functional analysis of these domains has been reported. We show here that various point mutations to KREPB4 and KREPB5 identify essential domains, and suggest that these proteins do not themselves perform RNase III catalysis. The zinc finger of KREPB4 but not KREPB5 is essential for editosome integrity and parasite viability, and mutation of the RNase III signature motif in KREPB5 prevents integration into editosomes, which is lethal. Isolated TAP-tagged KREPB4 and KREPB5 complexes preferentially associate with components of the deletion subcomplex, providing additional insights into editosome architecture. A new alignment of editosome RNase III sequences from several kinetoplastid species implies that KREPB4 and KREPB5 lack catalytic activity and reveals that the PUF motif is present in the editing endonucleases KREN1, KREN2, and KREN3. The data presented here are consistent with the hypothesis that KREPB4 and KREPB5 form intermolecular heterodimers with the catalytically active editing endonucleases, which is unprecedented among known RNase III proteins.

A protein-protein interaction map of trypanosome ~20S editosomes.

Mitochondrial mRNA editing in trypanosomatid parasites involves several multiprotein assemblies, including three very similar complexes that contain the key enzymatic editing activities and sediment at ~20S on glycerol gradients. These ~20S editosomes have a common set of 12 proteins, including enzymes for uridylyl (U) removal and addition, 2 RNA ligases, 2 proteins with RNase III-like domains, and 6 proteins with predicted oligonucleotide binding (OB) folds. In addition, each of the 3 distinct ~20S editosomes contains a different RNase III-type endonuclease, 1 of 3 related proteins and, in one case, an additional exonuclease. Here we present a protein-protein interaction map that was obtained through a combination of yeast two-hybrid analysis and subcomplex reconstitution with recombinant protein. This map interlinks ten of the proteins and in several cases localizes the protein region mediating the interaction, which often includes the predicted OB-fold domain. The results indicate that the OB-fold proteins form an extensive protein-protein interaction network that connects the two trimeric subcomplexes that catalyze U removal or addition and RNA ligation. One of these proteins, KREPA6, interacts with the OB-fold zinc finger protein in each subcomplex that interconnects their two catalytic proteins. Another OB-fold protein, KREPA3, appears to link to the putative endonuclease subcomplex. These results reveal a physical organization that underlies the coordination of the various catalytic and substrate binding activities within the ~20S editosomes during the editing process.

Nitrile-inducible gene expression in mycobacteria.

The ability to ectopically control gene expression is a fundamental tool for the study of bacterial physiology and pathogenesis. While many efficient inducible expression systems are available for Gram-negative bacteria, few are useful in phylogenetically distant organisms, such as mycobacteria. We have adapted a highly-inducible regulon of Rhodococcus rhodochrous to artificially regulate gene expression in both rapidly-growing environmental mycobacteria and slow-growing pathogens, such as Mycobacterium tuberculosis. We demonstrate that this artificial regulatory circuit behaves as a bistable switch, which can be manipulated regardless of growth phase in vitro, and during intracellular growth in macrophages. High-level overexpression is also possible, facilitating biochemical and structural studies of mycobacterial proteins produced in their native host.