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. The structural basis of both bacterial and eukaryotic translation is addressed 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 the past 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-level 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, each stalling a critical part of the machinery. Since the resolution of such density maps is often limited to 5–12 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.
Classification of Heterogeneous Samples
In many instances 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 since conformational and orientational variability are commingled in the experimental data set. Powerful classification methods based on maximum likelihood have now become available that can sort the data into groups without prior knowledge. Different conformational states can now be readily captured that coexist in the same sample, as demonstrated by the analysis of a sample containing a pretranslocational ribosome complex (Figure 1).
The classification software, notably the RELION program recently developed by Sjors Scheres (Medical Research Council, Cambridge), has helped us greatly in the ongoing studies of the eukaryotic translation initiation process, in collaboration with Tatyana Pestova and Christopher Hellen (SUNY Downstate Medical Center, Brooklyn). Typically, several eukaryotic initiation factors (eIFs) are involved, which are bound to the ribosome in different combinations, and only a small percentage (on the order of 5 percent) of the complexes contain the full set of components. This "needle in a haystack" problem was solved, leading to a reconstruction of the eukaryotic preinitation complex that shows eIF3, the helicase DHX29, and the ternary complex eIF2-Met-tRNA-GDPNP bound to the 40S ribosomal subunit (Figure 2).
Another example for the power of the new classification method is a preinitiation complex involving a hepatitis C virus (HCV)-related internal ribosome entry site (IRES) that takes over the host's translation machinery. In this instance, the molecular complex was found to exist in a continuous range of conformations (Figure 3).
EttA–Energy-Dependent Regulation of Translation in Bacteria
Cells express many ribosome-interacting factors whose functions and molecular mechanisms remain unknown. In a recent collaboration with John Hunt (Columbia University Department of Biological Sciences) and Ruben Gonzalez (Columbia University Department of Chemistry), we elucidated the mechanism of a newly characterized regulatory translation factor, energy-dependent translational throttle A (EttA), which is an Escherichia coli representative of the ATP-binding cassette F (ABC-F) protein family. Using cryo-EM, we showed that the ATP-bound form of EttA binds to the ribosomal transfer RNA (tRNA)-exit site, where it forms bridging interactions between the ribosomal L1 stalk and the tRNA bound in the peptidyl-tRNA–binding site. Using single-molecule fluorescence resonance energy transfer, we showed that the ATP-bound form of EttA restricts or abolishes ribosome and tRNA dynamics required for protein synthesis. This work is the first to determine the detailed molecular mechanism of an ABC-F family protein.
Getting Closer to the Atomic Level
Cryo-EM has been steadily gaining in resolution as a result of advances in instrumentation and data collection hardware and software. The highest published resolution we have obtained thus far is 5 Å, for the ribosome of Trypanosoma brucei (Figure 4). In this case, however, data collection was still performed on film, entailing the tedious task of scanning hundreds of electron micrographs. The advent of direct electron detection cameras is about to revolutionize the field. With the help of the Gatan K2 Summit camera, which is installed on our 300-kV Polara electron microscope, we now have several projects at the 4-Å resolution level, a resolution at which direct chain tracing starts to become feasible.
This work is being supported, in part, by grants from the National Institutes of Health.
As of February 10, 2014