Alternative conformations and motions adopted by 30S ribosomal subunits visualized by cryo-electron microscopy (original) (raw)
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J Mol Biol, 1999
A three-dimensional reconstruction of the 30 S subunit of the Escherichia coli ribosome was obtained at 23 A Ê resolution. Because of the improved resolution, many more structural details are seen as compared to those obtained in earlier studies. Thus, the new structure is more suitable for comparison with the 30 S subunit part of the 70 S ribosome, whose structure is already known at a better resolution. In addition, we observe relative and, to some extent, independent movements of three main structural domains of the 30 S subunit, namely head, platform and the main body, which lead to partial blurring of the reconstructed volume. An attempt to subdivide the data set into conformationally de®ned subsets reveals the existence of conformers in which these domains have different orientations with respect to one another. This result suggests the existence of dynamic properties of the 30 S subunit that might be required for facilitating its interactions with mRNA, tRNA and other ligands during protein biosynthesis.
Journal of Molecular Biology, 1999
A three-dimensional reconstruction of the 30 S subunit of the Escherichia coli ribosome was obtained at 23 Å resolution. Because of the improved resolution, many more structural details are seen as compared to those obtained in earlier studies. Thus, the new structure is more suitable for comparison with the 30 S subunit part of the 70 S ribosome, whose structure is already known at a better resolution. In addition, we observe relative and, to some extent, independent movements of three main structural domains of the 30 S subunit, namely head, platform and the main body, which lead to partial blurring of the reconstructed volume. An attempt to subdivide the data set into conformationally defined subsets reveals the existence of conformers in which these domains have different orientations with respect to one another. This result suggests the existence of dynamic properties of the 30 S subunit that might be required for facilitating its interactions with mRNA, tRNA and other ligands during protein biosynthesis.
RNA (New York, N.Y.), 2011
Four decades after early in vitro assembly studies demonstrated that ribosome assembly is a controlled process, our understanding of ribosome assembly is still incomplete. Just as structure determination has been so important to understanding ribosome function, so too will it be critical to sorting out the assembly process. Here, we used a viable deletion in the yjeQ gene, a recognized ribosome assembly factor, to isolate and structurally characterize immature 30S subunits assembled in vivo. These small ribosome subunits contained unprocessed 17S rRNA and lacked some late ribosomal proteins. Cryo-electron microscopy reconstructions revealed that the presence of precursor sequences in the rRNA induces a severe distortion in the 3' minor domain of the subunit involved in the decoding of mRNA and interaction with the large ribosome subunit. These findings suggest that rRNA processing events induce key local conformational changes directing the structure toward the mature assembly. ...
1999
During protein biosynthesis, ribosomes are believed to go through a cycle of conformational transitions. We have identi®ed some of the most variable regions of the E. coli 70S ribosome and its subunits, by means of cryo-electron microscopy and three-dimensional (3D) reconstruction. Conformational changes in the smaller 30S subunit are mainly associated with the functionally important domains of the subunit, such as the neck and the platform, as seen by comparison of heat-activated, non-activated and 50S-bound states. In the larger 50S subunit the most variable regions are the L7/L12 stalk, central protuberance and the L1-protein, as observed in various tRNA-70S ribosome complexes. Dierence maps calculated between 3D maps of ribosomes help pinpoint the location of ribosomal regions that are most strongly aected by conformational transitions. These results throw direct light on the dynamic behavior of the ribosome and help in understanding the role of these¯exible domains in the translation process. #
2012
Single-particle cryo-electron microscopy (cryo-EM) became a well-established method to study the structure and function of large macromolecular assemblies in a close to physiological environment. Cryo-EM reconstructions of ribosomal complexes trapped at different stages during translation, cotranslational targeting, and translocation provide new insights on a molecular level into these processes, which are vital for the correct localization and folding of all proteins in the cell. The EM structures in combination with biochemical experiments and available highresolution crystal or nuclear magnetic resonance (NMR) structures of individual factors and of the ribosome allow for interpretation in quasi-atomic detail of the molecular mechanism of ribosomal complexes, their conformational changes and dynamic interactions with factors like the signal recognition particle, SRP receptor, the translocon, and the chaperone trigger factor. The snapshots obtained by single-particle EM reconstructions enable us to follow the path of a nascent protein from the peptidyl-transferase center, through the ribosomal tunnel, to and across the translocon in the membrane. With new developments in image processing techniques it is possible to sort a biological homogenous sample into different conformational states and to reach subnanometer resolution such that folding of the nascent chain into secondary structure elements can be directly visualized. With improved cryo-electron tomography and correlative light microscopy and EM, it will be possible to visualize ribosomal complexes in their cellular context.
Cryo-electron microscopy as an investigative tool: the ribosome as an example
BioEssays, 2001
Cryo-electron microscopy allows the visualization of macromolecules in their native state. Combined with techniques of three-dimensional reconstruction, cryo-EM images of single molecules can be used to study macromolecular interactions. The ribosome, a large RNA±protein complex with multiple binding interactions, is an excellent test case illustrating the power of these new techniques. Conformational changes during the binding of tRNA and protein factors to the ribosome can now be studied without the interference of crystal packing. Now that the first X-ray structures of ribosomal subunits have become available, conformational changes observed by cryo-EM in different functional states can be traced back to internal rearrangements of the underlying structural framework. Electron microscopy, X-ray crystallography, and modeling should be used together in the endeavor to understand the functioning of the translational machinery. BioEssays 23:725±732, 2001.