Research in the Strobel laboratory focuses on biologically critical reactions catalyzed by RNA. Currently, the primary focus is on two systems: RNA splicing and ribosomal peptide bond formation. We utilize a multidisciplinary approach to these two ribozymes, including biochemistry, organic synthesis, enzyme kinetics, X-ray crystallography, and molecular biology.

Intron Splicing. The discovery of the RNA self-splicing group I intron provided the first demonstration that not all enzymes are proteins. We recently reported the X-ray crystal structure of a group I intron splicing intermediate trapped in the pre-exon ligation state [P.L. Adams et al. Nature 430, 45-50 (2004)]. The structure reveals how the intron uses unprecedented RNA motifs to select the 5'- and 3'-splice sites, and how it coordinates metal ions to promote the chemical reaction. This is the first splicing complex of any kind to include a complete intron, both exons and an organized active site occupied with metal ions. The exon ligation is chemically equivalent for pre-mRNA splicing by the spliceosome. As a result, the chemical themes of splice site selection, exon alignment, and catalytic metal ion positioning, which are manifest in this splicing intermediate complex, are likely to find parallels in pre-mRNA splicing. We are now undertaking several additional structural and biochemical studies to characterize the entire RNA splicing pathway. The overriding goals of these studies are to: (i) understand the mechanism of RNA splicing, (ii) explain how RNA tertiary structure is formed and active sites created in the absence of proteins, (iii) reveal how metal ions contribute to RNA catalysis, and (iv) visualize the nature of the transition state of the phosphoryl transfer reaction promoted during exon ligation.

Protein Synthesis. Crystallographic studies reveal that the ribosomal peptidyl transferase center is composed exclusively of rRNA, i.e., that the ribosome is a ribozyme. We aim to determine how this biologically fundamental reaction is catalyzed. We are taking several approaches to understand this enzyme, including: (i) synthesis and characterization of chiral transition state inhibitors to define the stereochemistry of the peptidyl transferase reaction; (ii) preparation of modified A-site and P-site tRNA substrates to test for substrate assisted catalysis of peptide bond formation by enzyme kinetic analysis; (iii) purification of mutant ribosomes to assess the role of rRNA functional groups; (iv) investigating the reaction transition state by kinetic isotope effect analysis and by determining the Bronsted coefficient for the ester aminolysis reaction.