Interdisciplinary Method Offers New Way To Study Ribozymes

May 04, 1998

CHAMPAIGN, Ill. -- An interdisciplinary approach recently demonstrated by chemists at the University of Illinois has opened a new avenue for spectroscopic and kinetic studies of metal-binding sites in ribonucleic acid enzymes (ribozymes).

"Ribozymes show great promise for gene therapy of viral diseases, such as AIDS, due to their ability to bind and cleave viral RNA targets with the help of metal ions," said Yi Lu, a U. of I. professor of chemistry. "Studying the structure of ribozymes is extremely important in understanding how they catalyze reactions."

While chemists are accustomed to probing the structures of metal-binding sites both in small inorganic complexes and in large metalloproteins, the study of ribozymes has proven difficult. In contrast to the high metal-binding affinity commonly found in protein active sites, the metal-binding affinity of the ribozyme active site is similar to that of the numerous structural metal-binding sites, making it difficult to distinguish between the two classes of sites.

"By pulling one trick each from the toolboxes of chemistry and biology, we have overcome this problem," Lu said. "Our method combines the high thiophilicity of mercury with the selective placing of phosphorothioates to increase the affinity at the active site and permit spectroscopic analysis."

In the April 29 issue of the Journal of the American Chemical Society, Lu and graduate students Lynette Cunningham and Jing Li reported the first spectroscopic study of the metal-binding site in a hammerhead ribozyme.

"Our results provide clear evidence for inner-sphere coordination of metal ions to the non-bridging sulfur at the cleavage site," Lu said. "More importantly, our results further show that this direct coordination is responsible for mercury's ability to support phosphorothioate ribozyme activity."

The phosphorothioate approach has been commonly employed in the study of enzyme reaction mechanisms. Several groups, including Tom Cech's and Olke Uhlenbeck's (both at the University of Colorado), used this approach to establish that ribozymes are metalloenzymes. Other groups, including Tom O'Halloran's at Northwestern University, have successfully demonstrated the use of mercury in metal ion derivatives for the spectroscopic study of metalloproteins.

"By combining the phosphorothioate approach with the metal ion derivative approach, we achieve two goals," Lu said. "First, the metal-binding affinity at the active site is increased dramatically without affecting the metal-binding affinity at other sites. Second, sulfur-metal specific spectroscopic signatures allow us to probe the active site of a ribozyme in solution."

The new methodology may allow chemists to probe the structure of ribozymes at a level of detail similar to that available for metalloproteins, Lu said.

University of Illinois at Urbana-Champaign

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