Faculty Profile: Steven T. Gregory, PhD

Steven T. Gregory
Steven T. Gregory, PhD
Associate Professor of Medical Science (Research)
Molecular Biology, Cell Biology, & Biochemistry
Work: +1 401-863-3652
The ribosome is the ribonucleoprotein particle responsible for protein synthesis in all cells. While ribosome functional sites are highly conserved, they are remarkably robust to mutation, withstanding substitutions at even universally conserved residues. Using genetics, structural biology and experimental evolution, we are investigating the ribosome's capacity to accommodate structural distortions into active conformations, and how such distortions can be ameliorated by compensatory evolution.

Institutions

Bu

Research Description

The ribosome is the central component of the universal protein synthesizing machinery, a highly cooperative 2.5 million Dalton RNA-protein complex that has been largely conserved throughout biological evolution. It is assumed that such conservation results from strong selective pressure against any structural changes which would have catastrophic consequences for function. Despite this conservation, the ribosome is surprisingly robust to mutation, capable of functioning with remarkable speed and accuracy even with base or amino acid substitutions at universally conserved residues. This paradox has yet to be resolved, in part because the structural consequences of such substitutions are not known in any detail. With the development of ribosome crystallography and genetic techniques, it is now possible to examine at high resolution the structural basis for the ribosome's robustness to mutation and address fundamental questions about RNA structure and ribosome evolution.

Mutational robustness of ribosome active sites
Many antibiotics interfere with protein synthesis by binding to ribosome functional centers, and antibiotic resistance can result from structural changes at these sites. Antibiotics thereby provide convenient genetic selections for targeting these highly conserved functional sites for mutation. We have developed the genetics of ribosomes of the bacterium Thermus thermophilus, the origin of a number of high-resolution ribosome crystal structures, and have identified many antibiotic-resistant mutants with base substitutions at conserved residues of either 16S or 23S rRNA. Such mutations create conformational distortions sufficient to influence ribosome-antibiotic interactions while maintaining ribosome structural integrity required for activity. In collaboration with the laboratories of Albert Dahlberg and Gerwald Jogl, we have begun to determine high-resolution crystal structures of these mutant ribosomes. Such experiments will allow us to ascertain how the ribosome accommodates structural changes into an active conformation and provide insights into the nature of the sequence conservation of ribosome active sites.

Compensatory evolution
Antibiotic-resistance mutations usually carry a substantial fitness cost, by virtue of their location in functionally important sites. This fitness cost implies that reversion to sensitivity should arise in populations after removal of antibiotic from the environment. Contrary to expectations, experimental evolution studies have shown that cultures of resistant mutants grown in the absence of antibiotics do not generally revert to sensitivity, but instead acquire compensatory mutations that improve fitness. Currently little is know about the nature of such compensatory mutations or the mechanism by which they improve fitness. To address these questions, we are applying an experimental evolution approach to identify compensatory mutations that restore fitness to antibiotic-resistant mutants of Thermus thermophilus. X-ray crystallography of such ribosomes is expected to explain the structural basis for fitness compensation. These experiments also have the potential to reveal previously unrecognized functional relationships between components of the ribosome.

Affiliations

Member, American Association for the Advancement of Science
Member, American Society for Microbiology

Funded Research

From the National Institutes of Health, 1 R01 GM094157-01, entitled "Structural Robustness of Ribosome Functional Centers", with Gerwald Jogl, Co-principle investigator. July 2010-June 2015

Selected Publications

  • Gregory, S.T., Demirci, H., Belardinelli, R., Monshupanee, T., Gualerzi, C., Dahlberg, A.E., and Jogl, G. (2009). Structural and functional studies of the Thermus thermophilus 16S rRNA methyltransferase RsmG. RNA 15:1693-1704. (2009)
  • Gregory, S.T., Carr, J.F. and Dahlberg, A.E. (2009). A signal relay between ribosomal protein S12 and elongation factor EF-Tu during decoding of mRNA. RNA 15:208-214. (2009)
  • Gregory, S.T. and Dahlberg, A.E. (2009). Genetic and structural analysis of base substitutions in the central pseudoknot of Thermus thermophilus 16S ribosomal RNA. RNA 15:215-223. (2009)
  • Demirci, H., Gregory, S.T., Dahlberg, A.E. and Jogl, G. (2007). Recognition of ribosomal protein L11 by the protein trimethyltransferase PrmA. EMBO Journal 26:567-577. (2007)
  • Carr, J.F., Hamburg, D.-M., Gregory, S.T., Limbach, P.A. and Dahlberg, A.E. (2006). Effects of streptomycin resistance mutations on posttranslational modification of ribosomal protein S12. Journal of Bacteriology 188:2020-2023. (2006)
  • Gregory, S.T., Carr, J.F., Rodriguez-Correa, D. and Dahlberg, A.E. (2005). Mutational analysis of 16S and 23S rRNA genes of Thermus thermophilus. Journal of Bacteriology 187:4804-4812. (2005)
  • Gregory, S.T. and Dahlberg, A.E. (2004). Peptide bond formation is all about proximity. Nature Structural and Molecular Biology 11:586-587. (2004)
  • Gregory, S.T., Cate, J.H. and Dahlberg, A.E. (2001). Streptomycin-resistant and streptomycin-dependent mutants of the extreme thermophile Thermus thermophilus. Journal of Molecular Biology 309:333-338. (2001)
  • Gabashvili, I.S., Gregory, S.T., Valle, M., Grassucci, R., Worbs, M., Wahl, M.C., Dahlberg, A.E. and Frank, J. (2001). The polypeptide tunnel system in the ribosome and its gating in erythromycin resistance mutants of L4 and L22. Molecular Cell 8:181-188. (2001)
  • Gregory, S.T. and Dahlberg, A.E. (1999). Erythromycin resistance mutations in ribosomal proteins L4 and L22 perturb the higher order structure of 23S ribosomal RNA. Journal of Molecular Biology 289:827-834. (1999)
  • Gregory, S.T. and Dahlberg, A.E. (1999). Mutations in the conserved P loop perturb the conformation of two structural elements in the peptidyl transferase center of 23S ribosomal RNA. Journal of Molecular Biology 285:1475-1483. (1999)