Structure-function relationships in RNA molecules: Studies of the malachite green aptamer and the signal recognition particle.

摘要

The function of an RNA molecule is inexorably linked to its structure, and the understanding of this link is critical for our knowledge of the biology of RNA. I have studied this relationship in two model systems, the malachite green aptamer and the signal recognition particle. In the first system I have solved the 2.8A crystal structure of the aptamer complexed with TMR, a high affinity ligand analog of malachite green. The ligand binding site is defined by an asymmetric internal loop flanked by a pair of helices. A U-turn and several non-canonical base interactions stabilize the folding of loop nucleotides around the TMR. The aptamer utilizes several tiers of stacked nucleotides arranged in pairs, triples, and a novel base quadruple to effectively encapsulate the ligand. In addition to the crystal structure, I have utilized stopped flow fluorescence spectroscopy to investigate the kinetics of folding of the aptamer. These studies show that the aptamer folding can be modeled by a simple two-state process from the unfolded/unbound state to the folded/bound state. alpha and phi kinetic analysis of mutant aptamers was used to elucidate the nature of the folding pathway of the aptamer core. In this pathway, the U-turn is almost completely formed in the transition state, while formation of the base quadruple, stacking of the gateway adenosines, and docking of the stacked adenosines into the minor groove of stem 2 occur later in the pathway. In the second system, I investigated the role of the central helix 5 of the signal recognition particle with regards to the translational stalling of signal sequence bearing transcripts. Our data suggest that helix 5 acts as a flexible but constraining linker between the two functional domains of the SRP, but that it does not rigidly fix their phase in relation to each other.