The aim of this book is to make available, to students and experts, a compendium of important articles covering (1) the development of single-article reconstruction techniques, and (2) the progress in the exploration of structural basis of translation in bacteria and eukaryotes by cryo-EM and single-particle reconstruction.
For novice and expert alike, a good overview of the development of tools and the ensuing discoveries spanning almost half a century has been lacking. Review articles attempting such an endeavor normally a very limited scope. The purpose of this book is to give the reader access to a selection of original articles spanning this period, from which he or she can form his or her opinion, the introductory text serving as a guide.
There is no competing book. The author has made pioneering contributions both in the development of the technique, which is now on the verge of attaining atomic resolution, and to the exploration of ribosome structure and function.
Selected reprints of key papers in the development of single-particle reconstruction methods, and in the research of ribosome structure and dynamics making use of these new methods. The reprints cover the period of 1969–2013.
Approaches to the recovery of three-dimensional information on a biological object, which are often formulated or implemented initially in an intuitive way, are concisely described here based on physical models of the object and the image-formation process. Both three-dimensional electron microscopy and X-ray tomography can be captured in the same mathematical framework, leading to closely-related computational approaches, but the methodologies differ in detail and hence pose different challenges. The editors of this volume, Gabor T. Herman and Joachim Frank, are experts in the respective methodologies and present research at the forefront of biological imaging and structural biology.
Supramolecular machines perform their work in the cell by going through many different states, distinguished by different conformations and free-energy levels. Ideally, in order to find out how these machines work, we would create a suitable in vitro environment containing all components including energy supply that allows the machine to function. We would then aim to take a “movie,” capturing their structure at highest resolution in a continuous fashion. Keeping within that film analogy, we might consider taking a large number of “snapshots” in equal small time intervals, each short enough, as in the macroscopic world, to eliminate jarring transitions. However, we would find out that this project has flaws both on the conceptual and the practical level. Conceptually, it is incorrect to equate a molecular machine’s progress to the workings of a macroscopic machine in motion since the states are not ordered in sequence of time but are visited in a stochastic manner, with occasional irreversible events such as NTP hydrolysis as the only mark of progress. In practical terms, there are in fact two problems, one affecting the way data for any given state can be captured, the other affecting the ability to obtain coverage of states in a continuum...
Cells express many ribosome-interacting factors whose functions and molecular mechanisms remain unknown. Here, we elucidate 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 demonstrate that the ATP-bound form of EttA binds to the ribosomal 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 show that the ATP-bound form of EttA restricts ribosome and tRNA dynamics required for protein synthesis. This work represents the first example, to our knowledge, in which the detailed molecular mechanism of any ABC-F family protein has been determined and establishes a framework for elucidating the mechanisms of other regulatory translation factors.
ABC-F proteins have evaded functional characterization even though they compose one of the most widely distributed branches of the ATP-binding cassette (ABC) superfamily. Herein, we demonstrate that YjjK, the most prevalent eubacterial ABC-F protein, gates ribosome entry into the translation elongation cycle through a nucleotide-dependent interaction sensitive to ATP/ADP ratio. Accordingly, we rename this protein energy-dependent translational throttle A (EttA). We determined the crystal structure of Escherichia coli EttA and used it to design mutants for biochemical studies including enzymological assays of the initial steps of protein synthesis. These studies suggest that EttA may regulate protein synthesis in energy-depleted cells, which have a low ATP/ADP ratio. Consistently with this inference, EttA-deleted cells exhibit a severe fitness defect in long-term stationary phase. These studies demonstrate that an ABC-F protein regulates protein synthesis via a new mechanism sensitive to cellular energy status.
Eukaryotic translation termination results from the complex functional interplay between two release factors, eRF1 and eRF3, in which GTP hydrolysis by eRF3 couples codon recognition with peptidyl-tRNA hydrolysis by eRF1. Here, we present a cryo-electron microscopy structure of pre-termination complexes associated with eRF1•eRF3•GDPNP at 9.7 -Å resolution, which corresponds to the initial pre-GTP hydrolysis stage of factor attachment and stop codon recognition. It reveals the ribosomal positions of eRFs and provides insights into the mechanisms of stop codon recognition and triggering of eRF3’s GTPase activity.
Hepatitis C virus (HCV) and classical swine fever virus (CSFV) messenger RNAs contain related (HCV-like) internal ribosome entry sites (IRESs) that promote 5′-end independent initiation of translation, requiring only a subset of the eukaryotic initiation factors (eIFs) needed for canonical initiation on cellular mRNAs. Initiation on HCV-like IRESs relies on their specific interaction with the 40S subunit which places the initiation codon into the P site, where it directly base-pairs with eIF2-bound initiator methionyl transfer RNA to form a 48S initiation complex. However, all HCV-like IRESs also specifically interact with eIF3, but the role of this interaction in IRES-mediated initiation has remained unknown. During canonical initiation, eIF3 binds to the 40S subunit as a component of the 43S pre-initiation complex, and comparison of the ribosomal positions of eIF3 and the HCV IRES revealed that they overlap, so that their rearrangement would be required for formation of ribosomal complexes containing both components. Here we present a cryo-electron microscopy reconstruction of a 40S ribosomal complex containing eIF3 and the CSFV IRES. Remarkably, although the position and interactions of the CSFV IRES with the 40S subunit in this complex are similar to those of the HCV IRES in the 40S–IRES binary complex, eIF3 is completely displaced from its ribosomal position in the 43S complex, and instead interacts through its ribosome-binding surface exclusively with the apical region of domain III of the IRES. Our results suggest a role for the specific interaction of HCV-like IRESs with eIF3 in preventing ribosomal association of eIF3, which could serve two purposes: relieving the competition between the IRES and eIF3 for a common binding site on the 40S subunit, and reducing formation of 43S complexes, thereby favouring translation of viral mRNAs.
Termination of messenger RNA translation in Bacteria and Archaea is initiated by release factors (RFs) 1 or 2 recognizing a stop codon in the ribosomal A site and releasing the peptide from the P-site transfer RNA. After release, RF-dissociation is facilitated by the G-protein RF3. Structures of ribosomal complexes with RF1 or RF2 alone or with RF3 alone—RF3 bound to a non-hydrolyzable GTP-analog—have been reported. Here, we present the cryo-EM structure of a post-termination ribosome containing both apo-RF3 and RF1. The conformation of RF3 is distinct from those of free RF3•GDP and ribosome-bound RF3•GDP(C/N)P. Furthermore, the conformation of RF1 differs from those observed in RF3-lacking ribosomal complexes. Our study provides structural keys to the mechanism of guanine nucleotide exchange on RF3 and to an L12-mediated ribosomal recruitment of RF3. In conjunction with previous observations, our data provide the foundation to structurally characterize the complete action cycle of the G-protein RF3.
Eukaryotic translation initiation begins with assembly of a 43S preinitiation complex. First, methionylated initiator methionine transfer RNA (Met-tRNAi
Met), eukaryotic initiation factor (eIF) 2, and guanosine triphosphate form a ternary complex (TC). The TC, eIF3, eIF1, and eIF1A cooperatively bind to the 40S subunit, yielding the 43S preinitiation complex, which is ready to attach to messenger RNA (mRNA) and start scanning to the initiation codon. Scanning on structured mRNAs additionally requires DHX29, a DExH-box protein that also binds directly to the 40S subunit. Here, we present a cryo-electron microscopy structure of the mammalian DHX29-bound 43S complex at 11.6 A°
resolution. It reveals that eIF2 interacts with the 40S subunit via its a subunit and supports Met-tRNAi Met in an unexpected P/I orientation (eP/I). The structural core of eIF3 resides on the back of the 40S subunit, establishing two principal points of contact, whereas DHX29 binds around helix 16. The structure provides insights into eukaryote-specific aspects of translation, including the mechanism of action of DHX29.
Molecular machines are workshops in the cell that bring molecules and substrates together in a
coordinated, processive way.1‐2. The molecular complexes acting as molecular machines are often
spatially localized and assembled in an ad‐hoc fashion, in response to the demands of the cell’s
metabolism. Products of such binding reactions can be RNA or DNA polymers, ATP, or other compounds
needed at the site. Well‐known examples are RNA and DNA polymerases, ATP synthase, the ribosome,
and the proteasome. Visualizing molecular machines in their various states of processing poses
challenges which traditional techniques of structure research find hard to meet. The complexes formed
in the course of the work cycle are typically large, relatively unstable, and flexible ‐‐ all properties that
disfavor X‐ray crystallography as a means of visualization. In addition, molecular machines can have
multiple binding partners and can go through numerous states, characterized by different
conformations and binding configurations. Purification of such complexes can be done through two
different routes: either from a cell extract or from an in vitro reconstituted system. In either case, it is
difficult to isolate the complex by biochemical means in one of its many states with the purity required
for structural research.
Cryo‐electron microscopy (cryo‐EM) is a fairly new technique for obtaining a 3D image of a
molecule from a large set of projections, harvested from micrographs of a field of molecules captured in
random orientations.3 In some cases, resolutions in the range of 4 to 5.5 Å have already been achieved
for the ribosome, an RNA‐containing molecular machine.4‐6 Purity of the sample in terms of
conformation and binding state of the molecule is evidently an absolute requirement for obtaining a
meaningful 3D image from the “single particle” projection data set. In certain cases, high purity can be
achieved by affinity imaging techniques.7‐9 However, because of inherent difficulties of finding
biochemical methods that guarantee 100% purity, it has been a goal in the development of cryo‐EM to
accomplish the purification of the molecules after the imaging, at the data processing step. Cryo‐EM
projection data have a typical signal‐to‐noise ratio in the order of 0.1 or even less.10 The challenge posed
by classification of such noisy data is compounded by the fact that variability stemming from a change in
viewing direction is intermingled with variability due to heterogeneity of composition or conformation.