Jamie H. D. Cate
Associate Professor of Biochemistry, Biophysics and Structural Biology*
*and of Chemistry
Molecular Basis for Protein Synthesis by the Ribosome
Protein synthesis is the universal mechanism for translating the genetic code into cellular function. The machine that carries out translation is the ribosome, a large RNA-protein complex whose structure is highly conserved in all kingdoms of life. Ribosomes, which are over 21 nm in diameter, interact with several different ligands and cofactors, including messenger RNA (mRNA), transfer RNA (tRNA), and proteins involved in the initiation, elongation, and termination of protein synthesis. Ribosomes are also dynamic entities; the small and large ribosomal subunits associate and dissociate during one full cycle of protein synthesis. We are exploring the process of protein synthesis by the ribosome. Key questions about the fundamental nature of translation remain unanswered. For example, how does the ribosome accurately move along an mRNA? And how do certain antibiotics, so useful in reducing infections, cripple ribosomes? In humans, how is translation regulated, and how do viruses circumvent these controls?
We are using x-ray crystallographic, biochemical, and biophysical approaches to unravel the mechanism of protein synthesis, both in bacteria and in humans. For example, we are probing the mechanism by which the ribosome translocates tRNAs from one binding site to the next after peptide bond formation. Many antibiotics degrade the accuracy of translation, block peptide bond formation, or prevent tRNA shuttling on the ribosome. We are also looking at the structure of the bacterial ribosome both in the absence and presence of these antibiotics to decipher their effects on protein synthesis. In addition we are studying how human pathogens such as the hepatitis C virus (HCV) avoids host defenses and hijacks the human ribosome to translate its own proteins.
X-ray crystal structures of the E. coli ribosome. We are using x-ray crystallography to probe the structural basis for the many aspects of protein biosynthesis that require the intact ribosome. Our goal is to make an atomic-resolution "movie" of a ribosome in the process of making a protein. X-ray crystallography provides the only available means to take atomic-resolution "snapshots" that will serve as frames in this movie. We have now obtained crystals of the entire E. coli ribosome that diffract x-rays to a resolution of 3.1-3.2 Å, and have determined structures of the ribosome in four states to a resolution of 3.5 Å. These structures have revealed the molecular basis for ribosomal subunit association and dynamics in unprecedented detail. They may also explain how the ribosome controls mRNA and tRNA substrate movement during translation. We are now extending the resolution of the structures to the limit of diffraction of the crystals. Moreover, these crystals provide an unprecedented opportunity to probe in atomic detail the effects of antibiotics on the full ribosome and mutations in the ribosome that lead to antibiotic resistance or perturb key steps in translation.
Translation initiation in humans. In eukaryotes, translation is initiated by a complex mechanism in which the 40S ribosomal subunit is recruited to the mRNA 5’-end cap structure, recognizes the start codon by a scanning mechanism before association with the 60S ribosomal subunit to form an active 80S ribosome. In an alternative pathway used by viruses such as the human pathogenic hepatitis C virus (HCV), an RNA structure called an internal ribosomal entry site (IRES) located in the 5’ untranslated region recruits, properly positions, and activates the ribosome in a cap- and end-independent process. Initiation from the HCV IRES involves only a small subset of the eukaryotic initiation factors (eIFs) needed for canonical translation initiation. We are studying aspects of both pathways of initiation using a variety of biochemical and structural approaches to provide a better understanding at the molecular level how these processes work.
Impact of molecular crowding on translation. Under physiological conditions, biochemical reactions occur in a crowded environment. Molecular crowding has its biggest impact on macromolecular interactions, including large conformational changes within a macromolecule. For example, molecular crowding dramatically influences protein folding pathways. In a bacterial cell, the concentration of macromolecules may reach one hundred times that in a typical in vitro biochemical reaction. Since molecular crowding is difficult to emulate outside of the cell, the function of biomolecules in crowded environments is not well understood. In protein biosynthesis, a number of conformational rearrangements in the ribosome have been identified from structural and hydrodynamic measurements. The impact of these rearrangements on the energetics of translation, especially in crowded cellular conditions, remains entirely unexplored.
We have recently devised new microfluidic systems for probing rapid biochemical kinetics in molecular crowding conditions. We are now using them to assess the effect of macromolecular crowding on protein synthesis. In particular, we are probing how the ribosome and elongation factor G convert the chemical energy of GTP hydrolysis into mechanical energy of mRNA and tRNA translocation. Our results will provide an entirely new perspective on the energetics and kinetics of translation as it occurs in the cell.
Liquid fuels produced from cellulosic biomass have the potential to reduce our dependence on oil. However, the current process for converting biomass to liquid fuels is too expensive for them to be cost competitive with conventional gasoline or diesel fuels. In the Cate lab, we study the biological degradation and utilization of cellulose by microorganisms. We believe a more complete understanding of microbial cellulose utilization will help to reduce the cost of cellulosic biofuels.
Cellulose is the most abundant biopolymer on earth and a promising feedstock for the production of biofuels. It is a highly crystalline, linear polysaccharide composed of beta-1-4 linked glucose molecules. The crystallinity makes cellulose resistant to hydrolysis. Fungi are ubiquitous degraders of cellulosic biomass, and are currently used as production hosts for commercial cellulase products.
In collaboration with Louise Glass and Michael Marletta's research groups, we are studying the degradation of cellulose by the filamentous fungus Neurospora crassa. Neurospora crassa is widely recognized as a model organism and has comprehensive genetic, biochemical, and functional genomic tools. We recently completed a system wide analysis of Neurospora's response to growth on cellulosic biomass and have identified a number of genes involved in the process (Tian and Beeson et al., PNAS, 2009). Ongoing work in the lab is focused on the biochemical characterization of these proteins and enzymes.
Munro, J.B., Altman, R.B., Tung, C.S., Cate, J.H., Sanbonmatsu, K.Y., Blanchard, S.C. (2010) Spontaneous formation of the unlocked state of the ribosome is a multistep process. PNAS, 107, 709-14.
Tian, C., Beeson, W.T., Iavarone, A.T., Sun J., Marletta, M.A., Cate, J.H. and Glass, N.L. (2009) Systems analysis of plant cell wall degradation by the model filamentous fungus Neurospora crassa. PNAS, 106, 22157-62.
Zhang, W., Dunkle, J.A. and Cate, J.H.D. (2009) Structures of the ribosome in intermediate states of ratcheting. Science, 325, 1014-7.
Borovinskaya, M.A., Shoji, S., Fredrick, K. and Cate, J.H.D. (2008) Structural basis for hygromycin B inhibition of protein biosynthesis. RNA, 14, 1590-9.
Pai, R.D., Zhang, W., Schuwirth, B.S., Hirokawa, G., Kaji, H., Kaji, A. and Cate, J.H.D. (2008) Structural Insights into ribosome recycling factor interactions with the 70S ribosome. J. Mol. Biol., 376, 1334-47.
Borovinskaya, M.A., Shoji, S., Holton, J.M., Fredrick, K. and Cate, J.H.D. (2007) A steric block in translation caused by spectinomycin. ACS Chem Biol, 2, 545-52.
Borovinskaya, M.A., Pai, R.D., Zhang, W., Schuwirth, B.S., Holton, J.M., Hirokawa, G., Kaji, H., Kaji, A. and Cate, J.H.D. (2007) Structural basis for aminoglycoside inhibition of bacterial ribosome recycling. Nat. Struct. Mol. Biol., 14, 727-32.
Berk, V., Zhang, W., Pai, R.D. and Cate, J.H.D. (2006) Structural basis for messenger RNA and transfer RNA positioning on the ribosome. PNAS 103, 15830-4.
Schuwirth, B.S., Day, J.M., Hau, C.W., Janssen, G.R., Dahlberg, A.E., Cate, J.H.D.† and Vila-Sanjurjo, A. (2006) Structural analysis of kasugamycin inhibition of translation. Nat. Struct. Mol. Biol., 13, 879-86.
Colón-Ramos, D.A., Shenvi, C.L., Weitzel, D.H., Gan, E.C., Matts, R., Cate, J. and Kornbluth, S. (2006) Direct ribosomal binding by a cellular inhibitor of translation. Nat. Struct. Mol. Biol. 13, 103-111.
Shenvi, C.L., Dong, K.C., Friedman, E.M., Hanson, J.A. and Cate, J.H.D. (2005) Accessibility of 18S rRNA in human 40S subunits and 80S ribosomes at physiological magnesium ion concentrations—Implications for the study of ribosome dynamics. RNA, 11, 1898-908.
Liau, A., Karnik, R., Majumdar, A. and Cate, J.H.D. (2005) Mixing crowded biological solutions in milliseconds. Analytical Chemistry, 77, 7618-25.
Schuwirth, B.S., Borovinskaya, M.A., Hau, C.W., Zhang, W., Vila-Sanjurjo, A., Holton, J.M. and Cate, J.H.D. (2005) Structures of the bacterial ribosome at 3.5 Å resolution. Science 310,827-834.
Last Updated 2010-07-23