Conformational dynamics of the Leptomonas collosoma spliced leader RNA

The spliced leader (SL) RNAs found in trypanosomes, nematodes and their relatives undergo a trans splicing reaction in which a short sequence called the spliced leader is attached to mRNAs made elsewhere in the genome. We have used a combination of physical and molecular biological techniques to probe the L. collosoma SL RNA structure.;A point mutational analysis by temperature jump, equilibrium melting experiments and ribonuclease mapping demonstrates that the SL RNA can exist in one of two conformational states which are readily separable on native polyacrylamide gels. The modification interference technique was applied to propose structures for two structural forms. The less stable form is that previously proposed, while the more stable form is a new structure that we have proposed. Mutational analysis also suggests that the region of the splice site is important for the tertiary structure, but has not indicated the nature of the tertiary structure.;Temperature jump experiments using DNA oligonucleotides probes complementary to regions which are single stranded in either of the two RNA forms demonstrate that the RNA can switch from one form to the other. A combination of temperature jump kinetics and stopped flow kinetics set an upper limit on the conformational switch at one second and also suggest that the switch takes place through a low activation energy pathway. The conformational switch may be important in the trans splicing mechanism in trypanosome cells.;The temperature jump time resolved thermal melting profile of the SL RNA shows two melting signals, one for tertiary structure and one for helical structure melting. Probing the RNA structure with a series of short, complementary DNA oligonucleotides demonstrates that the RNA structure is inconsistent with the model proposed using RNA folding programs. Further oligonucleotide structure probing of the RNA structure demonstrated that the 5...