THE SECONDARY STRUCTURE OF GUIDE RNA MOLECULES FROM TRYPANOSOMA-BRUCEI

RNA editing in kinetoplastid organisms is a mitochondrial RNA processi ng phenomenon that is characterized by the insertion and deletion of u ridine nucleotides into incomplete mRNAs. Key molecules in the process are guide RNAs which direct the editing reaction by virtue of their p rimary sequences in an RNA-RNA interaction with the pre-edited mRNAs. To understand the molecular details of this reaction, especially poten tial RNA folding and unfolding processes as well as assembly phenomena with mitochondrial proteins, we analyzed the secondary structure of f our different guide RNAs from Trypanosoma brucei at physiological cond itions. By using structure-sensitive chemical and enzymatic probes in combination with spectroscopic techniques we found that the four molec ules despite their different primary sequences, fold into similar stru ctures consisting of two imperfect hairpin loops of low thermodynamic stability. The molecules melt in two-state monomolecular transitions w ith T(m)s between 33 and 39 degrees C and transition enthalpies of -32 to -38 kcal/mol, Both terminal ends of the RNAs are single-stranded w ith the 3' ends possibly adopting a single-stranded, helical conformat ion. Thus, it appears that the gRNA structures are fine tuned to minim ize stability for an optimal annealing reaction to the pre-mRNAs while at the same time maximizing higher order structural features to permi t the assembly with other mitochondrial components into the editing ma chinery.