With the discovery nearly thirty years ago that RNA can catalyze reactions with proficiencies that approach those of protein enzymes, the central dogma of biology with RNA as a simple carrier molecule between DNA and proteins was overturned. Today RNA is recognized as an active catalyst in biology, in self-splicing of group I and group II introns, in various small ribozymes, and also as the catalytic center of the ribosome and spliceosome. These findings, and the fundamental ability of RNA to act both as an efficient information carrier and functional macromolecule led to proposal of an RNA World early in evolution.
We explore RNA catalysis to learn about the catalytic potential of RNA and to decipher what is fundamental to all biological catalysts through comparison with protein enzyme catalysis.
These studies also define the unique properties of RNA and proteins lead to catalytic and behavioral distinctions.
The fundamental properties and behaviors of RNA molecules that we uncover teach us about how the potential function of RNA early in evolution and about the function of RNA molecules in modern-day biology. This knowledge may also be applied as RNA is co-opted for medical, technological and industrial applications.
Energy from binding interactions can be used to facilitate reactions of bond substrates, a fundamental precept of enzymology posited by Jencks for protein enzymes and demonstrated in our studies of RNA enzymes.
We currently focus on the group I ribozyme, the most well-studied catalytic RNA in both structure and function. We harness previous studies, including multiple crystal structures, a robust phylogeny model, and a defined kinetic and thermodynamic framework for the Tetrahymena group I ribozyme, to delve more deeply into questions about catalytic RNA and, in particular, how an RNA scaffold can be used to sculpt an active site and how RNA achieves specific and strong molecular recognition.
We are also very interested in RNA conformational changes, as these transitions are key elements in nearly all RNA-mediated events.
Nearly all RNA-mediated events incolve conformational changes. We can learn about these transitions from studying catalysis and folding in model systems.
To answer these and additional questions, we use techniques including site-directed mutagenesis and site-specific chemical modifications to alter both the ribozyme itself and its substrates. The replacement of single functional groups within a complex RNA structure with multiple related functionalities is straightforward, whereas the corresponding replacements in proteins remain challenging. Function is probed via pre-steady state kinetics, and structure is probed using a battery of chemical footprinting approaches. Recent advances allow us to incorporate probes of local dynamics and single molecule fluorescence assays of functional conformational transitions as well as carry out high-throughput structure-function studies.
Finally, our collaboration with the Greenleaf lab allows us to now simultaneously probe thousands of mutants, allowing for the first time the depth and breadth of information needed to understand how an RNA scaffold established a functional active site.
1. Hougland, J., Piccirilli, J., Forconi, M., Lee, J., Herschlag, D. (2005) RNA World 3rd Edition How the Group I Intron Works: A Case Study of RNA Structure and Function. Gesteland, R.F., Cech, T.R., Atkins, J.F., Editors. Cold Spring Harbor Laboratory Press, New York. 133-205. (PDF File)
2. Narlikar, G.J., Herschlag, D. (1997) Annu. Rev. Biochem. 66, 19-59. Mechanistic Aspects of Enzymatic Catalysis: Lessons from Comparison of RNA and Protein Enzymes. PMID: 9242901. (Medline) (PDF File)
Some leading papers from the lab:
1. Shi, X.S., Bisaria, N., Benz-Moy, T., Bonilla, S., Pavlichin, D., Herschlag, D. (2014) J. Am. Chem. Soc. 136, 6643-6648. Roles of Long-range Tertiary Interactions in Limiting Dynamics of the Tetrahymena Group I Ribozyme. PMCID: PMC4021564. (Medline) (PDF File) (Supporting Info)
2. Benz-Moy, T.L., Herschlag, D. (2011) Biochemistry 50, 8733-8755. Structure-function Analysis from the Outside In: Long-range Tertiary Contacts in RNA Exhibit Distinct Catalytic Roles. PMCID: PMC3186870. (Medline) (PDF File) (Supporting Info)
3. Forconi, M., Sengupta, R.N., Liu, M-C., Sartorelli, A.C., Piccirilli, J.A., Herschlag, D. (2009) Angew. Chem. Int. Ed. 48, 7171-7175. Structure and Function Converge to Identify a Hydrogen Bond in the Group I Ribozyme Active Site. PMCID: PMC2862986. (Medline) (PDF File) (Supporting Info)
Selected as 'Hot Paper' by the Editors and highlighted in Nat. Chem. Biol. (2009) 5, 712.
4. Karbstein, K., Herschlag, D. (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 2300-2305. Extraordinarily Slow Binding of Guanosine to the Tetrahymena Group 1 Ribozyme. PMCID: PMC151335. (Medline) (PDF File) (Supporting Info)
5. Shan, S., Kravchuk, A.V., Piccirilli, J.A., Herschlag, D. (2001) Biochemistry 40, 5161-5171. Defining the Catalytic Metal Ion Interactions in the Tetrahymena Ribozyme Reaction. PMID: 11318638. (Medline) (PDF File) (Supporting Info)
6. Wang, S., Karbstein, K., Peracchi, A., Beigelman, L., Herschlag, D. (1999) Biochemistry 43, 14363-14378. Identification of the hammerhead ribozyme metal ion binding site responsible for rescue of the deleterious effect of a cleavage site phosphorothioate. PMID: 10572011. (Medline) (PDF File) (Supporting Info)
7. Narlikar, G.J., Herschlag, D. (1998) Biochemistry 37, 9902-9911. Direct Demonstration of the Catalytic Role of Binding Interactions in Enzymatic Reactions. PMID: 9665695. (Medline) (PDF File)
8. Hertel, K.J., Peracchi, A., Uhlenbeck, O.C., Herschlag, D. (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 8497-8502. Use of Intrinsic Binding Energy for Catalysis by an RNA Enzyme. PMCID: PMC22973. (Medline) (PDF File)
9. Peracchi, A., Beigelman, L., Usman, N., Herschlag, d. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 11522-11527. Rescue of Abasic Hammerhead Ribozymes by Exogenous Addition of Specific Bases. PMCID: PMC38090. (Medline) (PDF File)
10. Herschlag, D., Cech, T.R. (1990) Biochemistry 29, 10172-10180. Catalysis of RNA Cleavage by the Tetrahymena thermophilia Ribozyme. 2. Kinetic Description of the Reaction of an RNA Substrate that Forms a Mismatch at the Active Site. PMID: 2271646. (Medline) (PDF File)