The project I am working on focuses on structural studies of RNase H1. It is a sequence non-specific nuclease that degrades the RNA strand in RNA/DNA duplexes. It is involved in DNA replication and proliferation of retroviruses. We have solved crystal structures of Bacillus halodurans RNase H1 in complex with an RNA/DNA hybrid (Nowotny M, et al. Cell (121) 2005). Later, we also determined structures of the same enzyme in complex with the transition state mimic and the reaction product (Nowotny M. and Yang W. EMBO J. (25) 2006). Based on these snapshots of different stages of the catalysis we proposed a detailed mechanism by which RNase H hydrolyzes RNA. The movie below shows morphing of structures of RNase H1 in complex with the substrate (PDB: 1ZBI), transition state mimic (2G8F), and the product (2G8V).

Movie legend: The active site carboxylates are shown in green and the RNA in pink with phosphorus in yellow and backbone oxygens in red.Small spheres represent waters molecules and the larger purple spheres magnesium ions.The one on the right is termed magnesium ion A and the one on the left magnesium ion B.

The first stage of the movie is the prereactive state with scissile phosphate at the active site and metal ion A coordinating a nucleophile (water or hydroxide ion - small red sphere) positioned to attack the phosphorus of the scissile phosphate in SN2-type reaction. Note the unusual coordination of magnesium ion B with only five ligands and an irregular coordination.

Reaction proceeds with the movement of the metal ions closer together. They are now separated by 3.5, compared with 4.1 in the prereactive state. The movement of the metal ions is accompanied by a movement of the attacking nucleophile which promotes the formation of a pentavalent transition state. In this state the attacking hydroxide and leaving 3 -OH group are equidistant from the phosphorus atom and around 2.1 away. The atoms observed in the structure are shown in red and those modeled based on the known geometry of phosphoryl transfer transition state are in grey.

The next stage of the reaction is the breakage of the bond between the 3 -OH group and the phosphorus and the removal of liberated 5 -phosphate from the active site. One consequence of this movement is a change of metal ion B coordination from irregular to a regular octahedral one. This change from high-energy strained coordination to a lower energy relaxed one can provide the energy to drive the reaction. At the end of this stage the 5 -phosphate is stabilized by water-mediated interaction with the metal ion A. The last stage of the reaction (not shown) is the destabilization of these interactions to allow the dissociation of the product. This destabilization is likely carried out by a flexible residue from the active site of the protein.