Inline Orientation

The structure of the hairpin ribozyme with a 2' O-methyl nonreactive substrate analog is a good example of a ribozyme facilitating the in-line orientation of the 2' and 5' oxygens with the phosphorus (Rupert and Ferre-D'Amare

2001,2004; Ferre-D'Amare and Rupert 2002; Ferre-D'Amare 2004). Only small additional movement would be required to align the atoms to an in-line orientation for the reaction. In a recent structure of a prior-to-cleavage 'tethered' version of the hammerhead ribozyme, the 2'O-P5'O atoms are also approaching the in-line orientation (Murray et al. 2002; Dunham et al. 2003). In the precursor structures of the HDV ribozyme (Ke et al. 2004) the atoms involved in the reaction are not so favorably oriented. As noted above, the structure does reveal a dramatic sharp bend in the backbone at the scissile phosphate, and Ke et al. (2004) point out that there is room to rotate the nucleotide at the -1 position (5' to the cleavage-site phosphate) such that the necessary alignment could be attained.

While an active site that positions the groups for the in-line arrangement will facilitate the reaction, the magnitude of the effect is difficult to quantify. There is no HDV ribozyme-specific data that would address this issue. However, Soukup and Breaker (1999) in an insightful study, correlated the stability of phosphodiester bonds in RNA with the geometry of internucleotide linkage in RNAs of known structures. In addition to demonstrating a clear relationship of geometry to activity, they were able to estimate that the inline orientation could contribute as much as a 103-104-fold enhancement in cleavage rates relative to a linkage constrained in a non-inline orientation, or as little as a 10-20-fold enhancement relative to an unconstrained linkage.

0 0

Post a comment