Evidence for U-tail stabilization of gRNA/mRNA interactions in kinetoplastid RNA editing.

The most dramatic example of RNA editing is found in the mitochondria of trypanosomes. In these organisms, U-insertions/deletions can create mRNAs that are twice as large as the gene that encodes them. Guide RNAs (gRNAs) that are complementary to short stretches of the mature message direct the precise placements of the U residues. The binding of gRNA to mRNA is a fundamental step in RNA editing and understanding the relative importance of the elements that confer affinity and specificity on this interaction is critical to our understanding of the editing process. In this study, we have analyzed the relative binding affinities of two different gRNA/mRNA pairs. The affinity of gA6-14 for its message (ATPase 6) is high, with an apparent K(D) in the 5-10 nM range. In contrast, gCYb-558 has a low affinity for its cognate mRNA. Deletion of the gRNA U-tail caused a significant reduction in the binding affinity for only the gCYb-558 pair, and was observed only under physiological magnesium conditions. These results indicate that the U-tail contribution can differ substantially between the different gRNA/mRNA pairs. In addition, our results suggest that the efficiency of gRNA/mRNA interaction is highly dependent on thermodynamic parameters determined by the local sequences and their adopted structures surrounding the anchor-binding site.

In vitro nutritional requirements and metabolic products of pathogenic and nonpathogenic strains of Cryptobia salmositica: essential carbohydrates and amino acids.

Pathogenic and nonpathogenic strains of Cryptobia salmositica cultured in minimum essential medium (MEM) with several monosaccharides, disaccharides and amino acids were observed for differences in multiplication and motility. Metabolic end products (i.e. alanine, aspartate, carbon dioxide, lactate and pyruvate) were measured for logarithmically growing cells under aerobic conditions. The pathogenic strain of C. salmositica multiplied more readily in MEM supplemented with D(-)ribose, D(+)xylose, D(+)galactose, D(+)glucose, D(+)mannose and D(-)fructose. However, there were no significant differences in multiplication when the strains were cultured with the monosaccharide D(-)arabinose. The nonpathogenic strain multiplied significantly better than the pathogenic strain in the presence of the disaccharides alpha-lactose, maltose and sucrose. It also multiplied more readily when the amino acids L-glutamine and D(-)proline were added to MEM. The end products of carbohydrate catabolism under aerobic conditions were alanine, aspartate, carbon dioxide, lactate and pyruvate.