RNA Folding Pathways and Structural Motifs Revealed through Mechanistic and Spectroscopic Investigations of Group II Intron Ribozymes.
In contrast to the state of knowledge about protein folding, little is known about the pathways, the driving forces, or even the molecular interactions that stabilize RNA tertiary folding. Knowledge of the tertiary structure of RNA is critical for deconvoluting mechanisms of RNA catalysis, and understanding RNA recognition by other biochemical constituents. Particularly in the case of very large RNA molecules, studies of RNA folding are facilitated when the RNA has catalytic, or ribozyme activity. In that case, the RNA can report on effects of subtle structural alterations through deviations in ground-state and transition-state catalytic behavior. For many of these molecules, the tertiary architecture can be visualized from the formation of long-range Watson-Crick interactions that occur as strong phylogenetic covariations in base-sequence. Due to a relative paucity of conserved nucleotides and base covariations, the tertiary structure of group II self-splicing introns is believed to depend on the formation of alternative tertiary interactions, involving backbone, base and divalent metal ion functionalities. Therefore, the group II intron ribozymes provide an important model for exploring the variation in molecular interactions and driving forces that may be of general importance in RNA folding. Our investigations into this problem involve two separate approaches: one is based on enzymological analysis of structural alterations and the other involves real-time spectroscopic evaluation of changes in the folding pathway. We are using stopped-flow fluorescence to follow the course of group II intron core assembly. The most complicated core structural element is Domain 1 (D1) of the intron, which binds short oligonucleotides (substrates) analogous in sequence to 5'-exon sequences. These substrates are efficiently cleaved when chemically essential Domain 5 is presented together with D1. By analyzing the folding kinetics of wild-type D1 RNA, and comparing results with D1 deletion mutants, we have found that there is a series of two structural transitions that take place in D1, enabling it to bind a target sequence. These transitions play a crucial role in the specificity of group II intron sequence recognition. Once the target is bound, we have identified features responsible for fidelity control by group II ribozymes, enabling them to cleave at the proper site with high accuracy. By compiling results from studies such as these, we are attempting to build a dynamic structural model for assembly of core elements in group II intron ribozymes.