| Popis: |
Historically, all small catalytic RNAs have been shown to undergo global conformational changes upon phosphodiester cleavage. However, the most recent of numerous hepatitis delta virus (HDV) ribozyme crystal structures has challenged this trend, as this crystal structure suggests that the precursor structure is already product-like in conformation. To further investigate this unusual observation, we have extensively characterized the solution behavior of several three-stranded versions of the HDV ribozyme from the recent crystal structure. Fluorescence gel shift assays show that varying lengths of the 5'overhang sequence adjacent to the active site result in the same degree of cleavage, whereas noncleavable substrates exhibit significantly more heterogeneity. Complementary steady state and time-resolved FRET assays demonstrated that the length of the 5'overhang sequence adjacent to the cleavage site affects the rates of conformational change upon substrate binding and cleavage. Molecular dynamics (MD) simulations were also performed to gain insight into the atomic behavior and catalytic relevance of the HDV ribozyme from the Chen et al crystal structure. These simulations suggested that the dU-1dG1dG2 motif used in the crystal does not result in a catalytically fit ribozyme compared to an all-ribose construct. Furthermore, altered active site conditions also result in lowered catalytic fitness. These simulations suggested that catalytic fitness is greatly disrupted by deprotonation of C75, supporting the hypothesis of the role of C75 as a general acid. Simulations also showed that the magnesium ion resolved near the scissile phosphate results in favorable catalytic geometry compared to simulations neutralized with sodium. Our experimental results demonstrate that, despite previously published results, all forms of the HDV ribozyme undergo significant global conformational changes upon self-cleavage, and our simulations show that C75 is poised to act as a general acid during cleavage. |
