Skip Ribbon Commands Skip to main content
:

profile

Profile Image

Barbara L Golden

Biochemistry 

  • Professor of Biochemistry
765.496.6165
765.494.7897
BCHM Room 22

 Lab Members

Area of Expertise: Structural Basis for RNA function

RNA is not a passive messenger
In the post genomic age, non-coding RNA sequences (those that do not encode proteins) have been found unexpectedly to be very important for regulation of gene expression and other cellular processes. Many of these are functional RNAs that can fold into stable three-dimensional structures and thereby perform work in the cell. A wide variety of riboswitches, which toggle gene expression on and off, have been identified, and catalytic RNAs, or ribozymes, are being discovered in a variety of contexts, including eukaryotic transcriptomes. It is therefore essential to develop a detailed understanding of these systems in order to understand the catalytic and regulatory functions of ribozymes and riboswitches. In our laboratory, we undertake structural and mechanistic analyses of functional RNAs and ribozymes to provide a picture of how these molecules can work within the cell.

The HDV ribozyme has a hybrid engine
The hepatitis delta virus (HDV) ribozyme is a small ribozyme originally identified in the genome of the HDV. HDV is a human pathogen that co-infects with the hepatitis B virus and thereby leads to liver disease. The ribozyme self-cleaves viral RNAs, synthesized as tandem repeats during rolling circle replication, into genome-sized pieces. This ribozyme has evolved to function within human cells and therefore has the potential to be used as a therapeutic and can be used as a molecular biological tool. Recently, functional HDV-like ribozymes have been identified within the human transcriptome, in plants, fungi and insects, and in the mosquitoAnopheles gambiaewhere cleavage is activated in a developmentally-regulated fashion. Ribozyme self-cleavage thus represents a potential target for mosquito-borne pathogens, including Dengue virus, West Nile virus, and Malaria.

To create a snapshot of the ribozyme, we have trapped the molecule in a state prior to cleavage and solved its crystal structure. This gives us a picture of an RNA molecule poised to react. At the cleavage site, we observe an RNA nucleobase, cytosine 75 (C75), and a magnesium ion interacting with the cleavage site. This structure confirms a reaction mechanism in which C75 is initially protonated. We have shown that C75 possesses a dramatically shifted pK and can therefore participate in proton transfer reactions at neutral pH. This property allows it to donate the proton to the 5’-hydroxyl leaving group, thereby activating it for catalysis. Surprisingly, we saw a magnesium ion interacting with both the 2’-hydroxyl attacking group and the cleavage site phosphate to help position the substrate for cleavage and to activate the nucleophile. In the use of a magnesium ion in the cleavage reaction, the HDV ribozyme functions similarly to the group I introns. This mechanism represents a paradigm shift because the ribozymes, such as the HDV ribozyme, were thought to be too small to bind and position metals for Lewis acid catalysis, a mechanism in which the metal ion interacts directly with the 2’-hydroxyl nucleophile. By both positioning a metal ion and by shifting the pKof C75, the HDV ribozyme overcomes the intrinsic inertness of RNA. Thus, the HDV ribozyme is the first ribozyme that has been observed to use two distinct catalytic strategies to perform an RNA cleavage reaction.

These studies have provided new concepts in the understanding of RNA structure and RNA catalytic strategies that will aid in the understanding of other RNA catalysts. It is likely that the genomic sequencing enterprises will reveal new RNA catalysts that will either be widespread across evolution, or unique to individual organisms. Our work will aid in the process of identifying and characterizing novel ribozymes. 

Awards & Honors

(2011) Seed for Success Award. Purdue University.

(2000) PEW Scholar. The Pew Scholars Program in the Biomedical Sciences.

Selected Publications

Chen, J., Ganguly, A., Miswan, Z., Hammes-Schiffer, S., Bevilacqua, P. C., & Golden, B. L. (2013). Identification of the catalytic Mg2+ ion in the hepatitis delta virus ribozyme. Biochemistry, 52, 557-567. Retrieved from http://pubs.acs.org/doi/pdf/10.1021/bi3013092

Golden, B. L. (2013). Catalysis by RNA, structure themes: group I introns. In Encyclopedia of Biological Chemistry 2nd Edition (Vol. 3, pp. 397-400). Waltham, MA: Academic Press.

Golden, B. L., Hammes-Schiffer, S., Carey, P. R., & Bevilacqua, P. C. (2013). An integrated picture of HDV ribozyme catalysis. In Biophysics of RNA Folding (Vol. ., pp. 135-168). New York: Springer.

Golden, B. L. (2011). Two distinct catalytic strategies in the hepatitis delta virus ribozyme cleavage reaction. Biochemistry, 50, 9424-9433. Retrieved from pubs.acs.org/doi/pdf/10.1021/bi201157t

Veeraraghavan, N., Ganguly, A., Chen, J. H., Bevilacqua, P. C., Hammes-Schiffer, S., & Golden, B. L. (2011). Metal binding motif in the active site of the HDV ribozyme binds divalent and monovalent ions. Biochemistry, 50, 2672-2682. Retrieved from http://pubs.acs.org/doi/abs/10.1021/bi2000164

Chen, J. H., Yajima, R., Chadalavada, D. M., Chase, E., Bevilacqua, P. C., & Golden, B. L. (2010). A 1.9 Å crystal structure of the HDV ribozyme precleavage suggests both Lewis acid and general acid mechanisms contribute to phosphodiester cleavage. Biochemistry, 49, 6508-6518.

Kieft, J. S., Chase, E., Costantino, D. A., & Golden, B. L. (2010). Identification and Characterization of anion binding sites in RNA. RNA, 16, 1118-1123. Retrieved from http://rnajournal.cshlp.org/content/16/6/1118.long

Chen, J. H., Gong, B., Bevilacqua, P., Carey, P., & Golden, B. L. (2009). A Catalytic Metal Ion Interacts with the Cleavage Site G-U Wobble in the HDV Ribozyme. Biochemistry, 48(7), 1498-1507. Retrieved from http://pubs.acs.org/doi/abs/10.1021/bi8020108

Gong, B., Chen, Y., Christian, E. L., Chen, J. H., Chase, E., Chadalavada, D. M., . . . Carey, P. R. (2008). Detection of innersphere interactions between magnesium hydrate and the phosphate backbone of the HDV ribozyme using Raman crystallography. Journal of the American Chemical Society, 130(30), 9670-9672. Retrieved from http://pubs.acs.org/doi/abs/10.1021/ja801861s