The polypeptide tunnel system in the ribosome and its gating in erythromycin resistance mutants of L4 and L22.

Variations in the inner ribosomal landscape determining the topology of nascent protein transport have been studied by three-dimensional cryo-electron microscopy of erythromycin-resistant Escherichia coli 70S ribosomes. Significant differences in the mouth of the 50S subunit tunnel system visualized in the present study support a simple steric-hindrance explanation for the action of the drug. Examination of ribosomes in different functional states suggests that opening and closing of the main tunnel are dynamic features of the large subunit, possibly accompanied by changes in the L7/L12 stalk region. The existence and dynamic behavior of side tunnels suggest that ribosomal proteins L4 and L22 might be involved in the regulation of a multiple exit system facilitating cotranslational processing (or folding or directing) of nascent proteins.

Localization of the ribosomal protection protein Tet(O) on the ribosome and the mechanism of tetracycline resistance.

Tet(O) belongs to a class of ribosomal protection proteins that mediate tetracycline resistance. It is a G protein that shows significant sequence similarity to elongation factor EF-G. Here we present a cryo-electron microscopic reconstruction, at 16 A resolution, of its complex with the E. coli 70S ribosome. Tet(O) was bound in the presence of a noncleavable GTP analog to programmed ribosomal complexes carrying fMet-tRNA in the P site. Tet(O) is directly visible as a mass close to the A-site region, similar in shape and binding position to EF-G. However, there are important differences. One of them is the different location of the tip of domain IV, which in the Tet(O) case, does not overlap with the ribosomal A site but is directly adjacent to the primary tetracycline binding site. Our findings give insights into the mechanism of tetracycline resistance.

Hepatitis C virus IRES RNA-induced changes in the conformation of the 40s ribosomal subunit.

Initiation of protein synthesis in eukaryotes requires recruitment of the 40S ribosomal subunit to the messenger RNA (mRNA). In most cases, this depends on recognition of a modified nucleotide cap on the 5' end of the mRNA. However, an alternate pathway uses a structured RNA element in the 5' untranslated region of the messenger or viral RNA called an internal ribosomal entry site (IRES). Here, we present a cryo-electron microscopy map of the hepatitis C virus (HCV) IRES bound to the 40S ribosomal subunit at about 20 A resolution. IRES binding induces a pronounced conformational change in the 40S subunit and closes the mRNA binding cleft, suggesting a mechanism for IRES-mediated positioning of mRNA in the ribosomal decoding center.

Visualization of tRNA movements on the Escherichia coli 70S ribosome during the elongation cycle.

Three-dimensional cryomaps have been reconstructed for tRNA-ribosome complexes in pre- and posttranslocational states at 17-A resolution. The positions of tRNAs in the A and P sites in the pretranslocational complexes and in the P and E sites in the posttranslocational complexes have been determined. Of these, the P-site tRNA position is the same as seen earlier in the initiation-like fMet-tRNA(f)(Met)-ribosome complex, where it was visualized with high accuracy. Now, the positions of the A- and E-site tRNAs are determined with similar accuracy. The positions of the CCA end of the tRNAs at the A site are different before and after peptide bond formation. The relative positions of anticodons of P- and E-site tRNAs in the posttranslocational state are such that a codon-anticodon interaction at the E site appears feasible.

Three-dimensional cryo-electron microscopy localization of EF2 in the Saccharomyces cerevisiae 80S ribosome at 17.5 A resolution.

Using a sordarin derivative, an antifungal drug, it was possible to determine the structure of a eukaryotic ribosome small middle dotEF2 complex at 17.5 A resolution by three-dimensional (3D) cryo-electron microscopy. EF2 is directly visible in the 3D map and the overall arrangement of the complex from Saccharomyces cerevisiae corresponds to that previously seen in Escherichia coli. However, pronounced differences were found in two prominent regions. First, in the yeast system the interaction between the elongation factor and the stalk region of the large subunit is much more extensive. Secondly, domain IV of EF2 contains additional mass that appears to interact with the head of the 40S subunit and the region of the main bridge of the 60S subunit. The shape and position of domain IV of EF2 suggest that it might interact directly with P-site-bound tRNA.

Solution structure of the E. coli 70S ribosome at 11.5 A resolution.

Over 73,000 projections of the E. coli ribosome bound with formyl-methionyl initiator tRNAf(Met) were used to obtain an 11.5 A cryo-electron microscopy map of the complex. This map allows identification of RNA helices, peripheral proteins, and intersubunit bridges. Comparison of double-stranded RNA regions and positions of proteins identified in both cryo-EM and X-ray maps indicates good overall agreement but points to rearrangements of ribosomal components required for the subunit association. Fitting of known components of the 50S stalk base region into the map defines the architecture of the GTPase-associated center and reveals a major change in the orientation of the alpha-sarcin-ricin loop. Analysis of the bridging connections between the subunits provides insight into the dynamic signaling mechanism between the ribosomal subunits.

Haloarcula marismortui 50S subunit-complementarity of electron microscopy and X-Ray crystallographic information.

The large 50S subunit of the Haloarcula marismortui 70S ribosome was solved to 19 A using cryo-electron microscopy and single particle reconstruction techniques and to 9 A using X-ray crystallography. In the latter case, phases were determined by multiple isomorphous replacement and anomalous scattering from three heavy atom derivatives. The availability of X-ray and electron microscopy (EM) data has made it possible to compare the results of the two experimental methods. In the flexible regions of the 50S subunit, small differences in the mass distribution were detected. These differences can be attributed to the influence of packing in the crystal cell. The rotationally averaged power spectra of X-ray and EM were compared in an overlapping spatial frequency range from 60 to 13 A. The resulting ratio of X-ray to EM power ranges from 1 to 15, reflecting a progressively larger underestimation of the Fourier amplitudes by the electron microscope.

Major rearrangements in the 70S ribosomal 3D structure caused by a conformational switch in 16S ribosomal RNA.

Dynamic changes in secondary structure of the 16S rRNA during the decoding of mRNA are visualized by three-dimensional cryo-electron microscopy of the 70S ribosome. Thermodynamically unstable base pairing of the 912-910 (CUC) nucleotides of the 16S RNA with two adjacent complementary regions at nucleotides 885-887 (GGG) and 888-890 (GAG) was stabilized in either of the two states by point mutations at positions 912 (C912G) and 885 (G885U). A wave of rearrangements can be traced arising from the switch in the three base pairs and involving functionally important regions in both subunits of the ribosome. This significantly affects the topography of the A-site tRNA-binding region on the 30S subunit and thereby explains changes in tRNA affinity for the ribosome and fidelity of decoding mRNA.

EF-G-dependent GTP hydrolysis induces translocation accompanied by large conformational changes in the 70S ribosome.

Cryo-electron microscopy has been used to visualize elongation factor G (EF-G) on the 70S ribosome in GDP and GTP states. GTP hydrolysis is required for binding of all the domains of EF-G to the pretranslocational complex and for the completion of translocation. In addition, large conformational changes have been identified in the ribosome. The head of the 30S subunit shifts toward the L1 protein side, and the L7/L12 stalk becomes bifurcated upon EF-G binding. Upon GTP hydrolysis, the bifurcation is reversed and an arc-like connection is formed between the base of the stalk and EF-G.

Location of translational initiation factor IF3 on the small ribosomal subunit.

The location of translational initiation factor IF3 bound to the 30S subunit of the Thermus thermophilus ribosome has been determined by cryoelectron microscopy. Both the 30S.IF3 complex and control 30S subunit structures were determined to 27-A resolution. The difference map calculated from the two reconstructions reveals three prominent lobes of positive density. The previously solved crystal structure of IF3 fits very well into two of these lobes, whereas the third lobe probably arises from conformational changes induced in the 30S subunit as a result of IF3 binding. Our placement of IF3 on the 30S subunit allows an understanding in structural terms of the biochemical functions of this initiation factor, namely its ability to dissociate 70S ribosomes into 30S and 50S subunits and the preferential selection of initiator tRNA by IF3 during initiation.

Effect of buffer conditions on the position of tRNA on the 70 S ribosome as visualized by cryoelectron microscopy.

The effect of buffer conditions on the binding position of tRNA on the Escherichia coli 70 S ribosome have been studied by means of three-dimensional (3D) cryoelectron microscopy. Either deacylated tRNAfMet or fMet-tRNAfMet were bound to the 70 S ribosomes, which were programmed with a 46-nucleotide mRNA having AUG codon in the middle, under two different buffer conditions (conventional buffer: containing Tris and higher Mg2+ concentration [10-15 mM]; and polyamine buffer: containing Hepes, lower Mg2+ concentration [6 mM], and polyamines). Difference maps, obtained by subtracting 3D maps of naked control ribosome in the corresponding buffer from the 3D maps of tRNA.ribosome complexes, reveal the distinct locations of tRNA on the ribosome. The position of deacylated tRNAfMet depends on the buffer condition used, whereas that of fMet-tRNAfMet remains the same in both buffer conditions. The acylated tRNA binds in the classical P site, whereas deacylated tRNA binds mostly in an intermediate P/E position under the conventional buffer condition and mostly in the position corresponding to the classical P site, i. e. in the P/P state, under the polyamine buffer conditions.

Structure and structural variations of the Escherichia coli 30 S ribosomal subunit as revealed by three-dimensional cryo-electron microscopy.

A three-dimensional reconstruction of the 30 S subunit of the Escherichia coli ribosome was obtained at 23 A 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.

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.

Escherichia coli 70 S ribosome at 15 A resolution by cryo-electron microscopy: localization of fMet-tRNAfMet and fitting of L1 protein.

Cryo-electron microscopy of the ribosome in different binding states with mRNA and tRNA helps unravel the different steps of protein synthesis. Using over 29,000 projections of a ribosome complex in single-particle form, a three-dimensional map of the Escherichia coli 70 S ribosome was obtained in which a single site, the P site, is occupied by fMet-tRNAfMet as directed by an AUG codon containing mRNA. The superior resolution of this three-dimensional map, 14.9 A, has made it possible to fit the tRNA X-ray crystal structure directly and unambiguously into the electron density, thus determining the locations of anticodon-codon interaction and peptidyltransferase center of the ribosome. Furthermore, at this resolution, one of the distinctly visible domains corresponding to a ribosomal protein, L1, closely matches with its X-ray structure.

A 9 A resolution X-ray crystallographic map of the large ribosomal subunit.

The 50S subunit of the ribosome catalyzes the peptidyl-transferase reaction of protein synthesis. We have generated X-ray crystallographic electron density maps of the large ribosomal subunit from Haloarcula marismortui at various resolutions up to 9 A using data from crystals that diffract to 3 A. Positioning a 20 A resolution EM image of these particles in the crystal lattice produced phases accurate enough to locate the bound heavy atoms in three derivatives using difference Fourier maps, thus demonstrating the correctness of the EM model and its placement in the unit cell. At 20 A resolution, the X-ray map is similar to the EM map; however, at 9 A it reveals long, continuous, but branched features whose shape, diameter, and right-handed twist are consistent with segments of double-helical RNA that crisscross the subunit.

Cryoelectron microscopy and image analysis of the cardiac ryanodine receptor.

The three-dimensional structure of the cardiac muscle ryanodine receptor (RyR2) is described and compared with its skeletal muscle isoform (RyR1). Previously, structural studies of RyR2 have not been as informative as those for RyR1 because optimal conditions for electron microscopy, which require low levels of phospholipid, are destabilizing for RyR2. A simple procedure was devised for diluting RyR2 (in phospholipid-containing buffer) into a lipid-free buffer directly on the electron microscope grid, followed by freezing within a few seconds. Cryoelectron microscopy of RyR2 so prepared yielded images of sufficient quality for analysis by single particle image processing. Averaged projection images for RyR2, as well as for RyR1, prepared under the same conditions, were found to be nearly identical in overall dimensions and appearance at the resolution attained, approximately 30 A. An initial three-dimensional reconstruction of RyR2 was determined (resolution approximately 41 A) and compared with previously reported reconstructions of RyR1. Although they looked similar, which is consistent with the similarity found for the projection images, and with expectations based on the 66% amino acid sequence identity of the two isoforms, structural differences near the corners of the cytoplasmic assembly were observed in both two- and three-dimensional studies.

Purification and characterization of ryanodine receptor 3 from mammalian tissue.

The ryanodine receptors are intracellular Ca2+ release channels that play a key role in cell signaling via Ca2+. There are three isoforms. Isoform 1 from skeletal muscle and isoform 2 from heart have been characterized. Isoform 3 is widely distributed in many mammalian tissues although in minuscule amounts. Its low abundance has hampered its study. We now describe methodology to isolate mammalian isoform 3 in amounts sufficient for biochemical and biophysical characterization. Bovine diaphragm sarcoplasmic reticulum fractions enriched in terminal cisternae containing both isoforms 1 (>95%) and 3 (<5% of the ryanodine binding) served as starting source. Isoform 3 was selectively immunoprecipitated from the 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonic acid (CHAPS)-solubilized fraction and eluted with peptide epitope. Isoform 3 thus prepared is highly purified as characterized by SDS-polyacryamide gel electrophoresis, Coomassie Blue staining, and by high affinity ryanodine binding. The purified isoform 3 was incorporated into planar lipid bilayers, and its channel properties were studied. Channel characteristics in common with the other two isoforms are slope conductance, higher selectivity to Ca2+ versus K+ (PCa/K approximately 6), and response to drugs and ligands. In its response to Ca2+ and ATP, it more closely resembles isoform 2. The first two-dimensional structure of isoform 3 was obtained by cryoelectron microscopy and image enhancement techniques.

Visualization of elongation factor G on the Escherichia coli 70S ribosome: the mechanism of translocation.

During protein synthesis, elongation factor G (EF-G) binds to the ribosome and promotes the step of translocation, a process in which tRNA moves from the A to the P site of the ribosome and the mRNA is advanced by one codon. By using three-dimensional cryo-electron microscopy, we have visualized EF-G in a ribosome-EF-G-GDP-fusidic acid complex. Fitting the crystal structure of EF-G-GDP into the cryo density map reveals a large conformational change mainly associated with domain IV, the domain that mimics the shape of the anticodon arm of the tRNA in the structurally homologous ternary complex of Phe-tRNAPhe, EF-Tu, and a GTP analog. The tip portion of this domain is found in a position that overlaps the anticodon arm of the A-site tRNA, whose position in the ribosome is known from a study of the pretranslocational complex, implying that EF-G displaces the A-site tRNA to the P site by physical interaction with the anticodon arm.

Three-dimensional structure of the yeast ribosome.

The 80S ribosome from Saccharomyces cerevisiae has been reconstructed from cryo electron micrographs to a resolution of 35 A. It is strikingly similar to the 70S ribosome from Escherichia coli, while displaying the characteristic eukaryotic features familiar from reconstructions of ribosomes from higher eukaryotes. Aside from the elaboration of a number of peripherally located features on the two subunits and greater overall size, the largest difference between the yeast and E.coli ribosomes is in a mass increase on one side of the large (60S) subunit. It thus appears more elliptical than the characteristically globular 50S subunit from E.coli. The interior of the 60S subunit reveals a variable diameter tunnel spanning the subunit between the interface canyon and a site on the lower back of the subunit, presumably the exit site through which the nascent polypeptide chain emerges from the ribosome.