RNA editing in trypanosomatids is catalyzed by a high molecular mass RNP complex, which is only partially characterized. TbMP42 is a 42 kDa protein of unknown function that copurifies with the editing complex. The polypeptide is characterized by two Zn fingers and a potential barrel structure/OB-fold at its C terminus. Using recombinant TbMP42, we show that the protein can bind to dsRNA and dsDNA but fails to recognize DNA/RNA hybrids. rTbMP42 degrades ssRNA by a 3' to 5' exoribonuclease activity. In addition, rTbMP42 has endoribonuclease activity, which preferentially hydrolyzes non-base-paired uridylate-containing sequences. Gene silencing of TbMP42 inhibits cell growth and is ultimately lethal to the parasite. Mitochondrial extracts from TbMP42-minus trypanosomes have only residual RNA editing activity and strongly reduced endo-exoribonuclease activity. However, all three activities can be restored by the addition of rTbMP42. Together, the data suggest that TbMP42 contributes both endo- and exoribonuclease activity to the editing reaction cycle.

We have characterized the gene YOR347c of Saccharomyces cerevisiae and shown that it encodes a second functional pyruvate kinase isoenzyme, Pyk2p. Overexpression of the YOR347c/PYK2 gene on a multicopy vector restored growth on glucose of a yeast pyruvate kinase 1 (pyk1) mutant strain and could completely substitute for the PYK1-encoded enzymatic activity. PYK2 gene expression is subject to glucose repression. A pyk2 deletion mutant had no obvious growth phenotypes under various conditions, but the growth defects of a pyk1 pyk2 double-deletion strain were even more pronounced than those of a pyk1 single-mutation strain. Pyk2p is active without fructose-1,6-bisphosphate. However, overexpression of PYK2 during growth on ethanol did not cause any of the deleterious effects expected from a futile cycling between pyruvate and phosphoenolpyruvate. The results indicate that the PYK2-encoded pyruvate kinase may be used under conditions of very low glycolytic flux.Pyruvate kinase is the last enzyme in the glycolytic pathway of sugar catabolism. It catalyzes the irreversible conversion of phosphoenolpyruvate (PEP) into pyruvate by the addition of a proton and the loss of a phosphate group, which is transferred to ADP. Pyruvate kinases from a wide range of organisms have been extensively studied, and much is known about their physical and catalytic properties (8,35,38,39). Nearly all characterized eukaryotic pyruvate kinases are tightly regulated and are activated by fructose-1,6-bisphosphate (FBP). The mammalian muscle isoenzyme M1 is the only known pyruvate kinase that displays hyperbolic kinetics and lacks allosteric control (40). On the other hand, pyruvate kinases from prokaryotes can be activated by either FBP or other sugar phosphates (e.g., ribose phosphate), and those from trypanosomes can be activated by fructose-2,6-bisphosphate (35).In the yeast Saccharomyces cerevisiae, pyruvate kinase has been thought to be encoded solely by the PYK1 gene. The gene was cloned and sequenced by Burke et al. (16), and part of the nucleotide sequence was revised by McNally et al. (36). PYK1 codes for a 54.5-kDa protein (Pyk1p) consisting of 500 amino acids. Mutants defective in the PYK1 gene fail to grow on fermentable carbon sources and are even inhibited by them (17,18,30). However, they grow normally on ethanol or other gluconeogenic carbon sources. Under those conditions, hexose phosphates are provided by the gluconeogenic pathway, which uses the enzymes FBP and PEP carboxykinase to bypass the 6-phosphofructo-1-kinase and pyruvate kinase reactions, respectively. The concentrations of glycolytic metabolites in pyk1 deletion mutants after growth on an ethanol-containing medium are similar to the wild-type levels, but after addition of glucose, a large increase in the amounts of PEP and phosphoglycerates can be observed (12, 17). These results suggested that Pyk1p is the main enzyme which catalyzes the conversion of PEP into pyruvate in S. cerevisiae.The biochemical properties of yeast Pyk1p suggest that it plays a central regul...

RNA editing in African trypanosomes is characterized by a uridylate-specific insertion and/or deletion reaction that generates functional mitochondrial transcripts. The process is catalyzed by a multi-enzyme complex, the editosome, which consists of approximately 20 proteins. While for some of the polypeptides a contribution to the editing reaction can be deduced from their domain structure, the involvement of other proteins remains elusive. TbMP42, is a component of the editosome that is characterized by two C2H2-type zinc-finger domains and a putative oligosaccharide/oligonucleotide-binding fold. Recombinant TbMP42 has been shown to possess endo/exoribonuclease activity in vitro; however, the protein lacks canonical nuclease motifs. Using a set of synthetic gRNA/pre-mRNA substrate RNAs, we demonstrate that TbMP42 acts as a topology-dependent ribonuclease that is sensitive to base stacking. We further show that the chelation of Zn2+ cations is inhibitory to the enzyme activity and that the chemical modification of amino acids known to coordinate Zn2+ inactivates rTbMP42. Together, the data are suggestive of a Zn2+-dependent metal ion catalysis mechanism for the ribonucleolytic activity of rTbMP42.

Mitochondrial pre-messenger RNAs (pre-mRNAs) in African trypanosomes require RNA editing in order to mature into functional transcripts. The process involves the addition and/or removal of U nucleotides and is mediated by a high-molecular-mass complex, the editosome. Editosomes catalyze the reaction through an enzyme-driven pathway that includes endo/exoribonuclease, terminal uridylate transferase and RNA ligase activities. Here we show that editing involves an additional reaction step, a 3′ nucleotidyl phosphatase activity. The activity is associated with the editing complex and we demonstrate that the editosomal proteins TbMP99 and TbMP100 contribute to the activity. Both polypeptides contain endo-exonuclease-phosphatase domains and we show that gene ablation of either one of the two polypeptides is compensated by the other protein. However, simultaneous knockdown of both genes results in trypanosome cells with reduced 3′ nucleotidyl phosphatase and reduced editing activity. The data provide a rationale for the exoUase activity of the editosomal protein TbMP42, which generates nonligatable 3′ phosphate termini. Opposing phosphates at the two pre-mRNA cleavage fragments likely function as a roadblock to prevent premature ligation.

Our previous data indicated that GPM1 encodes the only functional phosphoglycerate mutase in yeast. However, in the course of the yeast genome sequencing project, two homologous sequences, designated GPM2 and GPM3, were detected. They have been further investigated in this work. Key residues in the deduced amino acid sequence, shown to be involved in catalysis for Gpm1 (i.e. His8, Arg59, His181) are conserved in both enzymes. Overexpression of the genes under control of their own promoters in a gpm1 deletion mutant did not complement for any of the phenotypes. This could in part be attributed to a lack of expression due to their weak promoters. Higher level expression under the control of the yeast PFK2 promoter partially complemented the gpm1 defects, without restoring detectable enzymatic activity. Nevertheless, deletion of either GPM2 or GPM3, or the two deletions in concert, did not produce any obvious lesions for growth on a variety of different carbon sources, nor did they change the levels of key intermediary metabolites. We conclude that both genes evolved from duplication events and that they probably constitute non‐functional homologues in yeast.