Faculty

We use a combination of approaches - including biochemistry, mechanistic enzymology, chemical biology, protein engineering and yeast genetics - to study 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 provided insight into the order of binding of ribosomal proteins to ribosomal RNA (rRNA) and has given a structural and thermodynamic rationale for this order. However, in eukaryotic cells the assembly process is much more complex, requiring a macromolecular machinery of >170 proteins and >70 RNAs. While we know that this machinery is absolutely essential, we have little understanding of the function of the individual players. By taking a biochemical approach to study these proteins, which is complimented by in vivo work in the yeast S. cerevisiae, we are pioneering the study of macromolecular function of these proteins. To tackle this fascinating problem we have focused on subcomplexes and their functions. Specific projects include:

1. Chemical Biology Tools for Dissecting Ribosome Assembly. To rapidly isolate unstable intermediates in ribosome assembly we will establish small-molecule control over ribosome biogenesis. Intermediates will then be purified and analyzed by mass spectrometry and RNA structure mapping. By analyzing distinct assembly intermediates we will establish a ribosome assembly map.

2. The Role of an Essential GTPase in Ribosome Assembly. GTPases act as molecular switches triggered by hydrolysis of GTP to GDP. Using mutagenesis and kinetic analysis we are dissecting in molecular detail how the switch operates in the essential ribosome assembly factor Bms1. In addition we are characterizing structurally and functionally a novel RNA binding site within Bms1.

3. 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 and characterize DEAD box proteins involved in a conformational switch that controls folding of the small ribosomal subunit.

Awards

Biological Scholar, 2006
Damon Runyon Postdoctoral Fellow, 2003
Boehringer Ingelheim Predoctoral Fellow, 1998
Heinrich Hertz Fellow, 1997

Representative Publications

1. Karbstein, K., "The Role of GTPases in Ribosome Assembly", Biopolymers, 2007, Sep., 1-11.

2. Karbstein, K., Lee, J. and Herschlag, D., "Probing the Role of a Secondary Structure Element at the 5'- and 3'-Splice Sites in Group I Intron Self-splicing: The Tetrahymena L-16 ScaI Ribozyme Reveals a New Role of the G.U Pair in Self-splicing", Biochemistry, 2007, 46, 4861-75.

1. Karbstein, K. and Doudna, J.A., "GTP-dependent Formation of a Ribonucleoprotein Subcomplex Required for Ribosome Biogenesis", J. Mol. Biol., 2006, 356, 432.

2. Karbstein, K., Jonas, S. and Doudna, J.A., "An essential GTPase promotes assembly of ribosomal processing complexes", Mol. Cell, 2005, 20, 497.

3. Karbstein, K., Tang, K.H. and Herschlag, D., "A Base Triple in the Tetrahymena Group I Core Affects the Reaction Equilibrium via a Threshold Effect", RNA, 2004, 10, 1730.

4. Karbstein, K. and Herschlag, D., "Extraordinarily Slow Binding of Guanosine to the Tetrahymena Group I Ribozyme: Implications for RNA Preorganization and Function", Proc. National Acad. Sciences, 2003, 100, 2300.

5. Wang, S.L., Karbstein, K., Peracchi, A., Biegelman, L. and Kerschlag, D., "Identification of the Hammerhead Ribozyme Metal Ion Binding Site that Rescues the Deleterious Effect of a Cleavage Site Phosphorothioate", Biochem., 1999, 38, 12363.