Analysis of the conformational switch involved in the replication of turnip crinkle virus satellite C RNA

Satellite C was used as a model system to study a proposed conformational switch involved in the replication of Turnip crinkle virus (TCV), a plus-strand virus. The satC RNA exists in a pre-active conformation that folds into an active conformation during the initiation of minus-strand synthesis. The active conformation in comparison to the pre-active conformation is well characterized and known to possess six secondary and primary elements on its 3' end. Attempts to characterize the pre-active satC structure using TGGE showed that it exists in one major form that denatured at 23.7°C. The stability of the structure was drastically increased in the presence of 10muM Mg2+, indicating the presence of long-range interactions on pre-active satC. Melting curves revealed that 17-20 base pair stacks were contributing to the 23.7°C main transition detected by TGGE. Analysis of select mutants showed that the 5' end and specific sequences like the derepressor (DR) sequence played important roles in maintaining the pre-active structure. Experimental data from the halves show them to fold independently of each other implying that the pre-active structure could form only if the halves are in a continuous sequence. The use of UV cross-linking confirmed the presence of long range interactions that were abrogated in the absence of the 5' terminal GG, reemphasizing the role of the 5' end. Structural calculations of satC by m-fold revealed an ensemble of structures that were subject to constraint application in order to shortlist potential pre-active and active candidates. Candidates consistent with the experimental data were isolated and a tentative model showing the switch from a pre-active structure to an active structure via the possible formation of intermediate structures has been developed.