Associate Professor of Chemistry, Biochemistry and Molecular Biology
email: jcate@lbl.gov
office: 708 B Stanley Hall
lab: 748 Stanley Hall
phone: 510.666.2749
fax: 510.666.2747
lab phone: 510.666.2748, 510.666.2693
Research Interests
Structural Biology and Biophysical Chemistry — The mechanisms of protein synthesis by the bacterial ribosome and regulation of translation in humans are being probed by x-ray crystallography, biophysical chemistry, and genetics.
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 20 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 read the genetic code? And how do certain antibiotics, so useful in reducing infections, cripple ribosomes?
We are using x-ray cyrstallographic, biochemical, and genetic approaches to unravel the mechanism of protein synthesis. To investigate translational fidelity, we are studying 70S ribosomal complexes trapped in the process of choosing the correct aminoacyl tRNA. We are also 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 or prevent tRNA shuttling on the ribosome. We are looking at the structure of the bacterial ribosome both in the absence and presence of these antibiotics to decipher their effects on protein synthesis.
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 three 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. We are in the process of determining structures of the ribosome in the presence of different classes of antibiotic.
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 microfluidics 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.
Biography
Associate Professor in Chemistry and Molecular and Cell Biology, born 1968. B.S. University of Denver (1990); M.S. University of Colorado (1994); Ph.D. Yale University (1997); Damon Runyon-Walter Winchell Postdoctoral Fellow, University of California, Santa Cruz (1998-1999); Associate Member, Whitehead Institute for Biomedical Research and Assistant Professor of Biology, MIT (1999-2001); Searle Scholar (2000-2003); Sloan Research Fellow (2006-2007).