Hermann T, Auffinger P, Scott W G, Westhof E
Institut de Biologie Moléculaire et Cellulaire du CNRS, 15 rue René Descartes, F-67084 Strasbourg, France.
Nucleic Acids Res. 1997 Sep 1;25(17):3421-7. doi: 10.1093/nar/25.17.3421.
In the presence of magnesium ions, cleavage by the hammerhead ribozyme RNA at a specific residue leads to 2'3'-cyclic phosphate and 5'-OH extremities. In the cleavage reaction an activated ribose 2'-hydroxyl group attacks its attached 3'-phosphate. Molecular dynamics simulations of the crystal structure of the hammerhead ribozyme, obtained after flash-freezing of crystals under conditions where the ribozyme is active, provide evidence that a mu-bridging OH-ion is located between two Mg2+ions close to the cleavable phosphate. Constrained simulations show further that a flip from the C3'- endo to the C2'- endo conformation of the ribose at the cleavable phosphate brings the 2'-hydroxyl in proximity to both the attacked phosphorous atom and the mu-bridging OH-ion. Thus, the simulations lead to a detailed new insight into the mechanism of hammerhead ribozyme cleavage where a mu-hydroxo bridged magnesium cluster, located on the deep groove side, provides an OH-ion that is able to activate the 2'-hydroxyl nucleophile after a minor and localized conformational change in the RNA.
在镁离子存在的情况下，锤头状核酶RNA在特定残基处的切割会产生2'3'-环磷酸酯和5'-OH末端。在切割反应中，一个被激活的核糖2'-羟基基团攻击其连接的3'-磷酸基团。对在核酶具有活性的条件下快速冷冻晶体后获得的锤头状核酶晶体结构进行分子动力学模拟，结果表明，一个μ-桥连OH离子位于靠近可切割磷酸基团的两个Mg2+离子之间。约束模拟进一步表明，可切割磷酸基团处的核糖从C3'-内向型构象翻转至C2'-内向型构象，会使2'-羟基靠近被攻击的磷原子和μ-桥连OH离子。因此，这些模拟为锤头状核酶切割机制带来了详细的新见解，即位于深沟一侧的一个μ-羟基桥连镁簇提供了一个OH离子，该离子能够在RNA发生微小的局部构象变化后激活2'-羟基亲核试剂。
