Crystal structure of a 70S ribosome-tRNA complex reveals functional interactions and rearrangements

Our understanding of the mechanism of protein synthesis has undergone rapid progress in recent years as a result of low-resolution X-ray and cryo-EM structures of ribosome functional complexes and high-resolution structures of ribosomal subunits and vacant ribosomes. Here, we present the crystal structure of the Thermus thermophilus 70S ribosome containing a model mRNA and two tRNAs at 3.7 A resolution. Many structural details of the interactions between the ribosome, tRNA, and mRNA in the P and E sites and the ways in which tRNA structure is distorted by its interactions with the ribosome are seen. Differences between the conformations of vacant and tRNA-bound 70S ribosomes suggest an induced fit of the ribosome structure in response to tRNA binding, including significant changes in the peptidyl-transferase catalytic site.     

Dissecting the structural features of β-arrestins as multifunctional proteins

β-arrestins bind active G protein-coupled receptors (GPCRs) and play a crucial role in receptor desensitization and internalization. The classical paradigm of arrestin function has been expanded with the identification of many non-receptor-binding partners, which indicated the multifunctional role of β-arrestins in cellular functions. To elucidate the molecular mechanism of β-arrestin-mediated signaling, the structural features of β-arrestins were investigated using X-ray crystallography and cryogenic electron microscopy (cryo-EM). However, the intrinsic conformational flexibility of β-arrestins hampers the elucidation of structural interactions between β-arrestins and their binding partners using conventional structure determination tools. Therefore, structural information obtained using complementary structure analysis techniques would be necessary in combination with X-ray crystallography and cryo-EM data. In this review, we describe how β-arrestins interact with their binding partners from a structural point of view, as elucidated by both traditional methods (X-ray crystallography and cryo-EM) and complementary structure analysis techniques.     

Applications of the molecular dynamics flexible fitting method

In recent years, cryo-electron microscopy (cryo-EM) has established itself as a key method in structural biology, permitting the structural characterization of large biomolecular complexes in various functional states. The data obtained through single-particle cryo-EM has recently seen a leap in resolution thanks to landmark advances in experimental and computational techniques, resulting in sub-nanometer resolution structures being obtained routinely. The remaining gap between these data and revealing the mechanisms of molecular function can be closed through hybrid modeling tools that incorporate known atomic structures into the cryo-EM data. One such tool, molecular dynamics flexible fitting (MDFF), uses molecular dynamics simulations to combine structures from X-ray crystallography with cryo-EM density maps to derive atomic models of large biomolecular complexes. The structures furnished by MDFF can be used subsequently in computational investigations aimed at revealing the dynamics of the complexes under study. In the present work, recent applications of MDFF are presented, including the interpretation of cryo-EM data of the ribosome at different stages of translation and the structure of a membrane-curvature-inducing photosynthetic complex.     

The Escherichia coli large ribosomal subunit at 7.5 A resolution

BACKGROUND: In recent years, the three-dimensional structure of the ribosome has been visualised in different functional states by single-particle cryo-electron microscopy (cryo-EM) at 13-25 A resolution. Even more recently, X-ray crystallography has achieved resolution levels better than 10 A for the ribosomal structures of thermophilic and halophilic organisms. We present here the 7.5 A solution structure of the 50S large subunit of the Escherichia coli ribosome, as determined by cryo-EM and angular reconstitution. RESULTS: The reconstruction reveals a host of new details including the long alpha helix connecting the N- and C-terminal domains of the L9 protein, which is found wrapped like a collar around the base of the L1 stalk. A second L7/L12 dimer is now visible below the classical L7/L12 'stalk', thus revealing the position of the entire L8 complex. Extensive conformational changes occur in the 50S subunit upon 30S binding; for example, the L9 protein moves by some 50 A. Various rRNA stem-loops are found to be involved in subunit binding: helix h38, located in the A-site finger; h69, on the rim of the peptidyl transferase centre cleft; and h34, in the principal interface protrusion. CONCLUSIONS: Single-particle cryo-EM is rapidly evolving towards the resolution levels required for the direct atomic interpretation of the structure of the ribosome. Structural details such as the minor and major grooves in rRNA double helices and alpha helices of the ribosomal proteins can already be visualised directly in cryo-EM reconstructions of ribosomes frozen in different functional states.     

RF3 induces ribosomal conformational changes responsible for dissociation of class I release factors

During translation termination, class II release factor RF3 binds to the ribosome to promote rapid dissociation of a class I release factor (RF) in a GTP-dependent manner. We present the crystal structure of E. coli RF3*GDP, which has a three-domain architecture strikingly similar to the structure of EF-Tu*GTP. Biochemical data on RF3 mutants show that a surface region involving domains II and III is important for distinct steps in the action cycle of RF3. Furthermore, we present a cryo-electron microscopy (cryo-EM) structure of the posttermination ribosome bound with RF3 in the GTP form. Our data show that RF3*GTP binding induces large conformational changes in the ribosome, which break the interactions of the class I RF with both the decoding center and the GTPase-associated center of the ribosome, apparently leading to the release of the class I RF.     

The structural changes in the ribosome during the elongation cycle

The plenty of data about structural changes in the ribosome during its functioning has been accumulated. The most interesting information on such changes was obtained by cryo-EM of various ribosomal complexes with the ligands and by combination of rRNA site-directed mutagenesis with the analysis of structural changes in ribosome by chemical modification technique (chemical probing). The most studied structural transformations of the ribosome interacting with tRNAs and elongation factors are considered in this review. The structural rearrangements are discussed in the context of interactions between the functional centers of the ribosome. We also describe the system of tertiary contacts between the rRNA helices and proteins which forms the universal structure in the ribosome. We pay attention that by means of such system the allosteric conformational signal can be transmitted between the functional centers. Besides the discussion of different biochemical data in the scope of structural data we also consider the hypothesis that the position of GTPase associated center (GAC) in the ribosome regulates the binding of elongation factors.     

Structural basis for interaction of the ribosome with the switch regions of GTP-bound elongation factors

Elongation factor G (EF-G) catalyzes tRNA translocation on the ribosome. Here a cryo-EM reconstruction of the 70S*EF-G ribosomal complex at 7.3 A resolution and the crystal structure of EF-G-2*GTP, an EF-G homolog, at 2.2 A resolution are presented. EF-G-2*GTP is structurally distinct from previous EF-G structures, and in the context of the cryo-EM structure, the conformational changes are associated with ribosome binding and activation of the GTP binding pocket. The P loop and switch II approach A2660-A2662 in helix 95 of the 23S rRNA, indicating an important role for these conserved bases. Furthermore, the ordering of the functionally important switch I and II regions, which interact with the bound GTP, is dependent on interactions with the ribosome in the ratcheted conformation. Therefore, a network of interaction with the ribosome establishes the active GTP conformation of EF-G and thus facilitates GTP hydrolysis and tRNA translocation.     

Ribosome recycling revisited

Ribosome recycling involves the coordinated action of the ribosome recycling factor (RRF), elongation factor EF-G and initiation factor IF3 to disassemble the post-termination complex, recycling the components for the next round of translation. The crystal structure of domain I of RRF (RRF-DI) in complex with the large ribosomal subunit from the eubacteria Deinococcus radiodurans at high resolution reveals the nature and details of the interactions between this protein factor and rRNA/protein components of the ribosome. Universally conserved arginine residues within the RRF-DI establish important interactions with nuleotides of the 23S rRNA, explaining why mutations at these positions abolish factor binding. Furthermore, in conjunction with cryo-EM reconstruction, the X-ray analysis provides a structural complement to the recent biochemical data, offering additional insight into the mechanism of ribosome recycling.     

Macromolecular electron microscopy in the era of structural genomics

Macromolecular machines carry out many cellular functions. Cryo-electron microscopy (cryo-EM) is emerging as a powerful method for studying the structure, assembly and dynamics of such macromolecules, and their interactions with substrates. With resolutions still improving, ‘single-particle’ analyses are already depicting secondary structure. Moreover, cryo-EM can be combined in several ways with X-ray diffraction to enhance the resolution of cryo-EM and the applicability of crystallography. Electron tomography holds promise for visualizing machines at work inside cells.     

Direct three-dimensional localization and positive identification of RNA helices within the ribosome by means of genetic tagging and cryo-electron microscopy

BACKGROUND: Ribosomes are complex macromolecular machines that perform the translation of the genetic message. Cryo-electron microscopic (cryo-EM) maps of the Escherichia coli 70S ribosome are approaching a resolution of 10 A and X-ray maps of the 30S and 50S subunits are now available at 5 A. These maps show a lot of details about the inner architecture of the ribosome and ribosomal RNA helices are clearly visible. However, in the absence of further biological information, even at the higher resolution of the X-ray maps many rRNA helices can be placed only tentatively. Here we show that genetic tagging in combination with cryo-EM can place and orient double-stranded RNA helices with high accuracy. RESULTS: A tRNA sequence inserted into the E. coli 23S ribosomal RNA gene, at one of the points of sequence expansion in eukaryotic ribosomes, is visible in the cryo-EM map as a peripheral 'foot' structure. By tracing its acceptor-stem end, the location of helix 63 in domain IV and helix 98 in domain VI of the 50S subunit could be precisely determined. CONCLUSIONS: Our study demonstrates for the first time that features of a three-dimensional cryo-EM map of an asymmetric macromolecular complex can be interpreted in terms of secondary and primary structure. Using the identified helices as a starting point, it is possible to model and interpret, in molecular terms, a larger portion of the ribosome. Our results might be also useful in interpreting and refining the current X-ray maps.     

Normal mode based flexible fitting of high-resolution structure into low-resolution experimental data from cryo-EM

A new method for the flexible fitting of high-resolution structures into low-resolution maps of macromolecular complexes from electron microscopy has been recently described in applications to simulated electron density maps. This method uses a linear combination of low-frequency normal modes in an iterative manner to deform the structure optimally to conform to the low-resolution electron density map. Gradient-following techniques in the coordinate space of collective normal modes are used to optimize the overall correlation coefficient between computed and measured electron densities. With this approach, multi-scale flexible fitting can be performed using all-atoms or Calpha atoms. In this paper, illustrative studies of normal mode based flexible fitting to experimental cryo-EM maps are presented for three different systems. Large, functionally relevant conformational changes for elongation factor G bound to the ribosome, Escherichia coli RNA polymerase and cowpea chlorotic mottle virus are elucidated as the result of the application of NMFF from high-resolution structures to cryo-electron microscopy maps.     

Ribosome recycling revisited

Ribosome recycling involves the coordinated action of the ribosome recycling factor (RRF), elongation factor EF-G, and the initiation factor IF3 to disassemble the posttermination complex, recycling the components for the next round of translation. The crystal structure of domain I of RRF (RRF-DI) in complex with the large ribosomal subunit from the eubacteria Deinococcus radiodurans at a high resolution reveals the nature and details of the interactions between this protein factor and the rRNA/protein components of the ribosome. Universally conserved arginine residues within the RRF-DI establish important interactions with nucleotides of the 23S rRNA, thus explaining why mutations at these positions abolish factor binding. Furthermore, in conjunction with cryo-EM reconstruction, the X-ray analysis provides a structural complement to the recent biochemical data, offering additional insight into the mechanism of ribosome recycling. Published in Russian in Molekulyarnaya Biologiya, 2006, Vol. 40, No. 4, pp. 742–750. The text was submitted by the authors in English.     

The SAXS solution structure of RF1 differs from its crystal structure and is similar to its ribosome bound cryo-EM structure

Bacterial class I release factors (RFs) are seen by cryo-electron microscopy (cryo-EM) to span the distance between the ribosomal decoding and peptidyl transferase centers during translation termination. The compact conformation of bacterial RF1 and RF2 observed in crystal structures will not span this distance, and large structural rearrangements of RFs have been suggested to play an important role in termination. We have collected small-angle X-ray scattering (SAXS) data from E. coli RF1 and from a functionally active truncated RF1 derivative. Theoretical scattering curves, calculated from crystal and cryo-EM structures, were compared with the experimental data, and extensive analyses of alternative conformations were made. Low-resolution models were constructed ab initio, and by rigid-body refinement using RF1 domains. The SAXS data were compatible with the open cryo-EM conformation of ribosome bound RFs and incompatible with the crystal conformation. These conclusions obviate the need for assuming large conformational changes in RFs during termination.     

Interaction of the G' domain of elongation factor G and the C-terminal domain of ribosomal protein L7/L12 during translocation as revealed by cryo-EM

During tRNA translocation on the ribosome, an arc-like connection (ALC) is formed between the G' domain of elongation factor G (EF-G) and the L7/L12-stalk base of the large ribosomal subunit in the GDP state. To delineate the boundary of EF-G within the ALC, we tagged an amino acid residue near the tip of the G' domain of EF-G with undecagold, which was then visualized with three-dimensional cryo-electron microscopy (cryo-EM). Two distinct positions for the undecagold, observed in the GTP-state and GDP-state cryo-EM maps of the ribosome bound EF-G, allowed us to determine the movement of the labeled amino acid. Molecular analyses of the cryo-EM maps show: (1) that three structural components, the N-terminal domain of ribosomal protein L11, the C-terminal domain of ribosomal protein L7/L12, and the G' domain of EF-G, participate in formation of the ALC; and (2) that both EF-G and the ribosomal protein L7/L12 undergo large conformational changes to form the ALC.     

Messenger RNA movement on the ribosome

Recent X-ray and cryo-EM studies of 70S ribosome complexes containing different types of messenger RNAs (mRNA) and transfer RNA (tRNA) have been reviewed. Changes of the mRNA path on the ribosome at initiation and elongation states have been described. Authors suggested, that the specific region of ribosomal 30S subunit ("platform") is a ribosome binding site of regulatory domains of mRNA which locates on the non-translated 5'-end of the mRNA.     

Neuronal RNA granules are ribosome complexes stalled at the pre-translocation state

The polarized cell morphology of neurons dictates many neuronal processes, including the axodendridic transport of specific mRNAs and subsequent translation. mRNAs together with ribosomes and RNA-binding proteins form RNA granules that are targeted to axodendrites for localized translation in neurons. It has been established that localized protein synthesis in neurons is essential for long-term memory formation, synaptic plasticity, and neurodegeneration. We have used proteomics and electron microscopy to characterize neuronal RNA granules (nRNAg) isolated from rat brain tissues or human neuroblastoma. We show that ribosome-containing RNA granules are morula-like structures when visualized by electron microscopy. Crosslinking-coupled mass-spectrometry identified a potential G3BP2 binding site on the ribosome near the eIF3d-binding site on the 40S ribosomal subunit. We used cryo-EM to resolve the structure of the ribosome-component of nRNAg. The cryo-EM reveals that predominant particles in nRNAg are 80S ribosomes, resembling the pre-translocation state where tRNA’s are in the hybrid A/P and P/E site. We also describe a new kind of principal motion of the ribosome, which we call the rocking motion.     

Interactions of the release factor RF1 with the ribosome as revealed by cryo-EM

In eubacteria, termination of translation is signaled by any one of the stop codons UAA, UAG, and UGA moving into the ribosomal A site. Two release factors, RF1 and RF2, recognize and bind to the stop codons with different affinities and trigger the hydrolysis of the ester bond that links the polypeptide with the P-site tRNA. Cryo-electron microscopy (cryo-EM) results obtained in this study show that ribosome-bound RF1 is in an open conformation, unlike the closed conformation observed in the crystal structure of the free factor, allowing its simultaneous access to both the decoding center and the peptidyl-transferase center. These results are similar to those obtained for RF2, but there is an important difference in how the factors bind to protein L11, which forms part of the GTPase-associated center of the large ribosomal subunit. The difference in the binding position, C-terminal domain for RF2 versus N-terminal domain for RF1, explains a body of L11 mutation studies that revealed differential effects on the activity of the two factors. Very recent data obtained with small-angle X-ray scattering now reveal that the solution structure of RF1 is open, as here seen on the ribosome by cryo-EM, and not closed, as seen in the crystal.     

Cryo-EM visualization of a viral internal ribosome entry site bound to human ribosomes: the IRES functions as an RNA-based translation factor

Internal initiation of protein synthesis in eukaryotes is accomplished by recruitment of ribosomes to structured internal ribosome entry sites (IRESs), which are located in certain viral and cellular messenger RNAs. An IRES element in cricket paralysis virus (CrPV) can directly assemble 80S ribosomes in the absence of canonical initiation factors and initiator tRNA. Here we present cryo-EM structures of the CrPV IRES bound to the human ribosomal 40S subunit and to the 80S ribosome. The CrPV IRES adopts a defined, elongate structure within the ribosomal intersubunit space and forms specific contacts with components of the ribosomal A, P, and E sites. Conformational changes in the ribosome as well as within the IRES itself show that CrPV IRES actively manipulates the ribosome. CrPV-like IRES elements seem to act as RNA-based translation factors.     

Consensus among flexible fitting approaches improves the interpretation of cryo-EM data

Cryo-elecron microscopy (cryo-EM) can provide important structural information of large macromolecular assemblies in different conformational states. Recent years have seen an increase in structures deposited in the Protein Data Bank (PDB) by fitting a high-resolution structure into its low-resolution cryo-EM map. A commonly used protocol for accommodating the conformational changes between the X-ray structure and the cryo-EM map is rigid body fitting of individual domains. With the emergence of different flexible fitting approaches, there is a need to compare and revise these different protocols for the fitting. We have applied three diverse automated flexible fitting approaches on a protein dataset for which rigid domain fitting (RDF) models have been deposited in the PDB. In general, a consensus is observed in the conformations, which indicates a convergence from these theoretically different approaches to the most probable solution corresponding to the cryo-EM map. However, the result shows that the convergence might not be observed for proteins with complex conformational changes or with missing densities in cryo-EM map. In contrast, RDF structures deposited in the PDB can represent conformations that not only differ from the consensus obtained by flexible fitting but also from X-ray crystallography. Thus, this study emphasizes that a "consensus" achieved by the use of several automated flexible fitting approaches can provide a higher level of confidence in the modeled configurations. Following this protocol not only increases the confidence level of fitting, but also highlights protein regions with uncertain fitting. Hence, this protocol can lead to better interpretation of cryo-EM data.