faculty

Assistant Professor of Chemistry
Ph.D., Stanford University

Biochemistry

Phone:  (734) 615 2867   
E-mail: kkarbst@umich.edu

Research Group

We use a combination of approaches - including biochemistry, mechanistic enzymology, protein engineering and yeast genetics - to study the complex biological process of eukaryotic ribosome assembly at the molecular level. Our ultimate goal is to understand the function of assembly factors, the order of events as well as the rationale for this order, aiming to delineate principles important for the assembly of other large RNA-protein complexes, such as the spliceosome or the signal recognition particle.

Ribosomes are large macromolecular machines that catalyze protein synthesis in all cells. Groundbreaking work in bacteria has led to insight into the order of binding of ribosomal proteins to ribosomal RNA (rRNA) and has also provided a structural and thermodynamic rationale for this order. However, in eukaryotic cells the assembly process is much more complex, requiring an enormous macromolecular machinery of > 170 proteins and > 70 RNAs. While we know that this machinery is absolutely essential for cell viability and ribosome assembly, we have little to no understanding of the function of the individual RNA or protein players. By taking a biochemical approach to study these proteins, which is complemented by in vivo work in the yeast S. cerevisiae , we are pioneering the study of the molecular function of these proteins. To tackle this fascinating, but complex biological problem, we have focused on subcomplexes and their functions. Specifically, we are interested in the following questions:

The Role of an Essential GTPase in Ribosome Assembly:

• Bms1 is a GTPase that regulates binding of the putative RNA processing factor Rcl1 to pre-ribosomes. GTPases act as molecular switches that undergo conformational changes upon hydrolysis of GTP to GDP. We are dissecting in molecular detail how the switch operates and how it is regulated using mutagenesis and kinetic and thermodynamic analysis.

• The GTPase Bms1 also binds an essential RNA molecule, called U3 snoRNA, which is suggested to act as a chaperone for rRNA folding. We are studying the RNA protein interaction by high-resolution structural analysis as well as probing individual interactions thermodynamically.

RNA Conformational Changes in Ribosome Assembly

• DEAD box proteins are ATP-dependent enzymes that catalyze unwinding of RNA structures and dissociation of RNA-binding proteins. They are ubiquitously involved in ribosome assembly, yet their function in this process remains unknown. We want to identify DEAD box proteins involved in binding and dissociation of U3 snoRNA to ribosomal RNA using a combination of genetics and biochemical experiments with purified components. Dissociation of U3 from ribosomal RNA likely represents a key folding step for ribosomal RNA and therefore represents a crucial step in ribosome assembly.

A Multiprotein Complex Involved in Cytoplasmic Maturation of 40S Ribosomes

• Cytoplasmic maturation of 40S ribosomes requires methylation of conserved adenines, an endonucleolytic cleavage step, as well as binding of a few ribosomal proteins.   Yet, proteins required for this process include also include a protein kinase and an ATPase, suggesting that this step is intricately regulated. We are studying the functions of these and other proteins involved in 40S maturation with a goal of dissecting this regulated multi-step assembly process at the molecular level.

Chemical Biology Tools for Dissecting Ribosome Assembly

• To isolate intermediates in ribosome assembly we will generate conditional mutants of ribosome assembly proteins that can be rapidly rendered nonfunctional. One approach in the design of such proteins is to separate functionally important subdomains of a protein and then control their interaction using a small molecule, such as rapamycin. By fusing the rapamycin-binding proteins to our protein of interest, Bms1, we will create a mutant protein that requires the presence of rapamycin for function. This mutant will be used to accumulate intermediates during ribosome assembly and will be characterized by proteomic analysis, including mass spectrometry. A similar strategy will be carried for other proteins that also function at specific regulatory points during ribosome biogenesis.

AWARDS

• UM Biological Scholar
• Damon Runyon Postdoctoral Fellow (2003)
• Boehringer Ingelheim Predoctoral Fellow (1998)
• Heinrich Hertz Fellow (1997)

REPRESENTATIVE PUBLICATIONS

1. Karbstein, K., and Doudna, J. A. (2006) GTP-dependent Formation of a Ribonucleoprotein Subcomplex Required for Ribosome Biogenesis. Journal of Molecular Biology 356, 432-443
   
2. Karbstein, K., Jonas, S. and Doudna, J. A. (2005) An essential GTPase promotes assembly of ribosomal processing complexes. Molecular Cell 20, 633-643. [Preview by Dutca & Culver Molecular Cell 20,497-499.]
   
3. Karbstein, K., and Doudna, J. A. (2004) RNA: Primed for Packing? Chemistry & Biology 11, 149-151.
   
4. Karbstein, K., Tang, K. H. and Herschlag, D. (2004) A Base Triple in the Tetrahymena Group I Core Affects the Reaction Equilibirum via a Threshold Effect. RNA 10, 1730-1739.
   
5. Karbstein, K.,and Herschlag, D. (2003) Extraordinarily Slow Binding of Guanosine to the Tetrahymena Group I Ribozyme: Implications for RNA Preorganization and Function. Proceedings of the National Academy of Sciences 100, 2300-2305.
   
6. Wang, S.L., Karbstein, K., Peracchi, A., Beigelman, L., and Herschlag, D. (1999) Identification of the Hammerhead Ribozyme Metal Ion Binding Site that Rescues the Deleterious Effect of a Cleavage Site Phosphorothioate. Biochemistry 38, 14363-14378.