DIFFERENT TRYPANOSOMA-BRUCEI GUIDE RNA MOLECULES ASSOCIATE WITH AN IDENTICAL COMPLEMENT OF MITOCHONDRIAL PROTEINS IN-VITRO

kRNA editing is a mitochondrial transcript maturation process which ev olved in kinetoplastid protozoa. It entails the insertion and deletion of exclusively uridine nucleotides directed by gRNAs into pre-mRNAs. Other participating components are not currently known. The aim of thi s study was to identify mitochondrial proteins that are in direct phys ical contact with gRNAs thereby possibly involved in the editing react ion. At low monovalent cation concentration (30 mM KCI) 8 polypeptides with apparent molecular weights ranging from 124 to 9 kDa specificall y cross-linked to gRNAs. Three of the proteins, 90, 21, and 9 kDa in s ize, were able to bind at higher salt concentrations (greater than or equal to 100 mM) indicating an enhanced affinity to the gRNA molecules . No cross-links were identified at greater than or equal to 250 mM KC I. Four gRNAs, specific for different editing domains of the ATPase 6 and ND7 pre-mRNAs, were in contact with the same set of mitochondrial polypeptides suggesting the assembly of an identical RNP complex that does not include pre-mRNA molecules. The binding of the 90 kDa protein was sensitive to the presence of U-nucleotides at the 3'-end of the g RNAs and could specifically be blocked by modifying free sulfhydryl gr oups. The interaction with the 124 kDa polypeptide was inhibited by va nadyl ribonucleosides, implicating a role for 2', 3' hydroxyl groups i n the gRNA - protein interaction.