Professor of MCD Biology
Robert L. Sinsheimer Prof. of Molecular Biology
Director, Center for Molecular Biology of RNA
A.B., University of California, Berkeley
Ph.D., University of Oregon
1965-66 Natl Inst of Health Postdoctoral Fellow,
MRC Laboratory of Molecular Biology, Cambridge
1966-68 Natl Inst of Health Postdoctoral Fellow, Inst of Molecular Biology, Univ of Geneva, Switzerland
Ribosome Structure and Function
Ribosomes are complex molecular machines that are responsible for carrying out protein synthesis – translation of the genetic code. Their structures are highly conserved, and fundamentally similar in all organisms. A major question is, why do ribosomes contain such large amounts of RNA (50-60% of their mass)? It has gradually become clear that ribosomal RNA itself is centrally involved in translation, and may be a relic of the RNA World, a period of early molecular evolution when it is proposed that RNA carried out the genomic and catalytic roles of DNA and protein.
The Noller laboratory studies ribosome structure and function using a wide range of approaches, including X-ray crystallography, chemical probing methods, molecular genetics, comparative sequence analysis, fluorescence resonance energy transfer (FRET), including the use of single-molecule methods. The ultimate goal of these studies is to understand how the ribosome works at the molecular level: what are the moving parts of the machine, and how do they move in three dimensions to enable translation?
X-ray Crystal Structure of the Ribosome
We have recently solved the structure of the whole 70S ribosome from Thermus thermophilus at a resolution of 3.7 Angstroms. This has enabled us to describe all of the detailed molecular interactions of the ribosome, including ones involving the ribosomal RNAs, ribosomal proteins, tRNAs and mRNA. This provides a molecular basis for beginning to address the important functional questions. Our next goals for crystallography are to solve the structures of the ribosome trapped in different intermediate states of translation, including complexes containing elongation factors and other components.
Movement Inside the Translational Engine
On the basis of chemical footprinting results, we realized that the tRNAs move on the ribosome during the translocation step of protein synthesis in two steps: first they move at their acceptor CCA ends on the large ribosomal subunit, and then they move at their anticodon ends on the small subunit. We believe that both steps are catalyzed by the elongation factor EF-G, but the second step depends on GTP hydrolysis. We want to understand how the interactions between EF-G and the ribosomal machinery results in movement of the tRNAs and mRNA during translation. Clues from structural studies and from biochemical experiments implicate certain molecular features of ribosomal RNA (but also ribosomal proteins) in this movement. We are using chemical probing, FRET and mutational alteration of RNA and proteins to address these questions.
Functional Interactions of Translational Factors with the Ribosome
During protein synthesis, the ribosome interacts with initiation factors, elongation factors, release factors and ribosome recycling factor. Little is understood about their molecular interactions with the ribosome, and what they actually do to help the ribosome through the translational cycle. We are using chemical footprinting and directed hydroxyl radical probing to position the factors on the ribosome, and fluorescence methods to catch them in action. We are also attempting to crystallize complexes of the ribosome bound to the factors. Since most factor-catalyzed functions can be carried out by the ribosome itself under certain conditions, it is likely that these are all fundamentally mechanisms of the ribosome, whose rates and accuracy are increased by the factors.
Korostelev A, Trakhanov S, Laurberg M, Noller HF. Crystal structure of a 70S ribosome-tRNA complex reveals functional interactions and rearrangements. Cell. 126 (6):1065-77 (2006).
Fredrick, K. and Noller, H.F. Catalysis of ribosomal translocation by sparsomycin. Science 300(5622): 1159-62 (2003).
Lancaster L., Kiel, M.C., Kaji, A., and Noller, H.F. Orientation of Ribosome Recycling Factor in the Ribosome from Directed Hydroxyl Radical Probing. Cell 111: 129-140 (2002).
Fredrick, K. and Noller, H.F. Accurate Translocation of mRNA by the Ribosome Requires a Peptidyl Group or Its Analog on the tRNA Moving into the 30S P Site. Mol Cell 9: 1125-31 (2002).
Dallas, A. and Noller, H.F. Interaction of Translation Initiation Factor 3 with the 30S Ribosomal Subunit. Mol Cell 8: 855-64 (2001).
Thompson, J., Kim, D.F., O'Connor, M., Lieberman, K., Bayfield, M.A., Gregory, S.T., Green, R., Noller, H.F., and Dahlberg, A.E. Analysis of mutations at residues A2451 and G2447 of 23S rRNA in the peptidyl transferase active site of the 50S ribosomal subunit. PNAS 98: 9002-7 (2001).
Noller, H.F., Yusupov, M., Yusupova, G., Baucom, A., Lieberman, K., Lancaster, L., Dallas, A., Fredrick, K., Earnest, T.N., and Cate, J.H.D. 2001. Structure of the Ribosome at 5.5 Å Resolution and Its Interactions with Functional Ligands. Cold Spring Harbor Symposia on Quantitative Biology: The Ribosome, Volume 66. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, pp. 57-66.
Yusupova, G., Yusupov, M., Cate, J.H.D., and Noller, H.F. The Path of Messenger RNA Through the Ribosome. Cell 106: 233-241 (2001).
Yusupov, M., Yusupova, G., Baucom, A., Lieberman, K., Earnest, T. N., Cate, J. H., and Noller, H. F. Crystal Structure of the Ribosome at 5.5 Å Resolution. Science 292: 883-896 (2001).
Wilson, K.S., Ito, K., Noller, H.F., and Nakamura, Y., Functional sites of interaction between release factor RF1 and the ribosome. Nat Struct Biol 7: 866-70 (2000).
Cate, J.H.,Yusupov, M.M., Yusupova, G. Zh., Earnest, T.N. and Noller, H.F. X-ray crystal structures of 70S ribosome functional complexes. Science 285: 2095-2104 (1999).
Culver, G.M., Cate, J.H., Yusupova, G.Zh., Yusupov, M. M. and Noller, H.F. Identification of an RNA-protein bridge spanning the ribosomal subunit interface. Science 285: 2133-2135 (1999).