My group studies translation, the process by which the genetic message carried by mRNA is translated into a polypeptide chain, which then folds into a working protein. Both bacterial and eukaryotic translation are targeted in these studies. Our primary tool is visualization of in vitro complexes of the translating ribosome by cryo-electron microscopy (cryo-EM) combined with single-particle reconstruction, a method of structure research developed in my lab over the course of 30 years.
Molecules are rapidly frozen so that they are trapped in a close-to-native state in a thin (~1,000-Å) layer of vitreous ice. In contrast to x-ray crystallography, which requires highly ordered crystals, cryo-EM and single-particle reconstruction can produce a three-dimensional density map from freestanding macromolecules. This is done by combining tens of thousands of images showing different views of the macromolecule that exists in as many copies in the sample. In 1998 such a map, obtained by my group from a sample of the Haloarcula marismortui ribosome, was used by Thomas Steitz (HHMI, Yale University) in the phasing of his x-ray data, which two years later resulted in the first atomic structure of a bacterial 50S subunit.
This powerful method also makes it possible to analyze the motions and dynamic changes of "molecular machines" (assemblies of dynamically interacting molecules that perform one of the cell's functions, such as transcription or translation) during their functional cycle. If all assemblies are present in the same conformational state, then the density map produced by three-dimensional reconstruction can be interpreted as a meaningful "3D snapshot" of the macromolecular machine along its dynamic course. To get every ribosome in the sample "in lockstep," one uses some type of intervention: antibiotics, GTP nonhydrolyzable analogs, or targeted mutations, all affecting a critical part of the machinery. Since the resolution of such density maps is currently limited to 6–12 Å, sophisticated methods of docking and flexible fitting are required to interpret each map in terms of the underlying atomic structures available from x-ray crystallography.
We have recently made significant progress in three aspects of the technology, all leading to advances in the elucidation of translation. First, in collaboration with José María Carazo (Centro Nacional de Biotechnología, Madrid), we have advanced the methodology for sorting images of heterogeneous molecules into homogeneous subpopulations. Second, we have improved the cryo-EM resolution of a particularly stable ribosomal complex to 6.7 Å. Third, we have collaborated with Klaus Schulten (University of Illinois at Urbana-Champaign) in the development of a novel method for flexible fitting based on molecular dynamics.
Classification of Heterogeneous Samples
Often the sample contains the molecular machine in two or more different conformational or binding states, despite efforts of biochemical purification. These projection images must be sorted by conformation, which is difficult because conformational and orientational variability are commingled in the experimental data set. In collaboration with Carazo's group, we have developed a powerful classification method based on maximum likelihood. In contrast to conventional supervised classification techniques used in single-particle reconstruction, which require the knowledge of reference structures, this new technique requires a minimum of prior knowledge (Figure 1).
Progress in Visualization
The 6.7-Å map of the Escherichia coli ribosome is shown in Figure 2. The complex visualized here is "frozen" in a state just after aminoacyl-tRNA has entered the ribosome in complex with elongation factor Tu (EF-Tu) and GTP, and after GTP has been hydrolyzed. The antibiotic kirromycin was used to stall the complex in the GDP state. The map shows an extraordinary degree of molecular detail directly comparable to the x-ray structures of the ribosome and EF-Tu. At some places along the A-form rRNA helices, phosphorus atoms are actually visible as "bumps," indicating that some of the structural information reaches the 5-Å range. Both α-helices and β-sheets can be identified by appearance and by reference to the x-ray structures. The new map, interpreted in terms of the atomic structure, has allowed us to characterize the interactions between EF-Tu, GDP, and the ribosomal contact sites of the factor. It also has led to the discovery of a hydrophobic gating mechanism underlying GTP hydrolysis on EF-Tu during the decoding process.
Flexible Fitting
Our atomic interpretation of the aforementioned 6.7-Å density map was the result of a collaboration with Schulten's group that has led to the development of a general tool for flexible fitting, termed molecular dynamics flexible fitting, or MDFF. Here molecular dynamics simulation is used with an added force-field term whose purpose is to drive the structure locally into the cryo-EM map. (Secondary structure reinforcement is imposed to prevent the structure from violating stereochemistry). Thus, after initial rigid-body alignment of the x-ray structure with the density map, the computational procedure "molds" the atomic structure into this map, yielding an atomic interpretation of the observed conformational changes.
Significant progress has also been achieved in our efforts to study the mechanism of mRNA-tRNA translocation. Following our discovery of the ratchet motion, which is a large-scale conformational change of the ribosome (involving a rotation of the small versus large subunit) essential for translocation, several groups have studied and further characterized this motion, using bulk or single-molecule fluorescence resonance energy transfer. These studies found that at physiological Mg2+ concentrations, the ratchet motion of the pretranslocational ribosome (i.e., after the transfer of the peptide bond) occurs spontaneously, requiring no EF-G.
In collaboration with Rachel Green (HHMI, Johns Hopkins University) we have used cryo-EM and classification to investigate such a sample. We found at least two distinct conformations and tRNA-binding configurations, distinguished by the presence or absence of the ratchet motion and by the positions of the tRNAs (classical versus hybrid; Figure 3). A recent reanalysis of the same data set with an improved classification method resulted in the discovery of a well-populated intermediate state, as well.
The discovery of spontaneous ratcheting dovetails nicely with our earlier observation that binding of all factors, with the exception of EF-Tu, induces the ratchet motion. The list of factors includes IF2, EF-G, RF3, and RRF. We had found earlier, in collaboration with Charles Brooks III (then at the Scripps Research Institute), that the ratchet motion is a natural consequence of the ribosome's architecture, as it is a principal mode of the structure's dynamic response to a mechanical perturbation. Together the findings imply that the ribosome performs several unrelated tasks by channeling energy from the thermal environment to achieve a conformational change that is productive for the respective task. This also implies that the ratcheted ribosome, not the classic conformation as previously thought, is the binding target of EF-G∙GTP.
The study of the structural basis for translation and translational regulation in eukaryotes, which is a new frontier of research, is still handicapped by the lack of a crystal structure. However, cryo-EM maps have been obtained for the ribosomes of several organisms, including yeast and human, and these can be used as a basis for building atomic models that capitalize on high sequence homology between bacterial and eukaryotic ribosomes in functionally important core regions. We have now used the density map that we had obtained earlier for a close relative to yeast to guide the construction of a comprehensive model (Figure 4), which should have general utility in the interpretation of future cryo-EM studies of eukaryotic translation.
This work was supported, in part, by grants from the National Science Foundation and the National Institutes of Health.
As of May 30, 2012
