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Lambert, Dominic

Microbiology and Molecular Genetics

2005

Ph. D.

This dissertation describes the use of photocrosslinking, kinetics and computer-assisted molecular modeling to investigate the formation of catalytically active structures of the hairpin and hammerhead ribozymes. Metal ions facilitate the folding of the hairpin ribozyme, but do not participate directly in catalysis. The ion metal complex cobalt (III) hexaammine supports folding and activity of the ribozyme and also mediates specific internucleotide photocrosslinks, several of which retain catalytic ability. These crosslinks imply that the active core structure organized by cobalt hexaammine differs distinctly from that mediated by magnesium and that revealed in the crystal structure. Residues U+2 and C+3 of the substrate, in particular, adopt different conformations in cobalt hexaammine. U+2 is bulged out of loop A and stacked on residue G36, whereas the nucleotide at position +3 is stacked on G8, a nucleobase crucial for catalysis. Cleavage kinetics performed with +2 variants and a C+3 U variant correlate with the crosslinking observations. Variants that decreased cleavage rates in magnesium up to 70-fold showed only subtle decreases or even increases in observed rates when assayed in [Co(NH₃)₆]³⁺. We propose a model of the [Co(NH₃)₆]³⁺-mediated catalytic core generated by MC-SYM that is consistent with these data. Recently, we presented evidence that the hammerhead ribozyme forms a compact structure in which the two residues flanking the cleavage site approach and stack upon two guanosines (G8 and G12). In the crystal structure of the hammerhead, the cleavable linkage is at least 10-15Å from these guanosines. Here we report similar observations made in a native form of the hammerhead ribozyme. Although many of the crosslinks isolated were found to be inactive, we obtained a cobalt hexaammine-dependent crosslink from a pyrrolocytidine at position 17 to residue G 12 that retained catalytic activity. Moreover, we characterized multiple crosslinks that suggest a structural rearrangement in the U-turn, positioning residue G5 in the vicinity of position 1.1. We also observed intriguing spontaneous crosslinks triggered by nucleotide analogues at positions distant from the crosslinked residues according to the crystal structure. Among others, a 6- thioG5 induced a spontaneous crosslink between residues G12 and C17, suggesting the proximity of these three nucleobases. These findings confirm that that the proximity of the cleavable linkage to nucleobases G8 and G12 is intrinsic to all hammerheads and not only to minimal constructs, and provide additional evidence that the crystal structures depict an inactive ground state that must undergo a significant conformational change to assemble a functional active site. Here, we present a model of the active fold that rationalizes much of the new and historical experimental data on the hammerhead.