Anat Bashan

Structure of Functionally Activated Small Ribosomal Subunit at 3.3 Å Resolution

The small ribosomal subunit performs the decoding of genetic information during translation. The structure of that from Thermus thermophilus shows that the decoding center, which positions mRNA and three tRNAs, is constructed entirely of RNA. The entrance to the mRNA channel will encircle the message when a latch-like contact closes and contributes to processivity and fidelity. Extended RNA helical elements that run longitudinally through the body transmit structural changes, correlating events at the particles far end with the cycle of mRNA translocation at the decoding region. 96% of the nucleotides were traced and the main fold of all proteins was determined. The latter are either peripheral or appear to serve as linkers. Some may assist the directionality of translocation.

High Resolution Structure of the Large Ribosomal Subunit from a Mesophilic Eubacterium

We describe the high resolution structure of the large ribosomal subunit from Deinococcus radiodurans (D50S), a gram-positive mesophile suitable for binding of antibiotics and functionally relevant ligands. The over-all structure of D50S is similar to that from the archae bacterium Haloarcula marismortui (H50S); however, a detailed comparison revealed significant differences, for example, in the orientation of nucleotides in peptidyl transferase center and in the structures of many ribosomal proteins. Analysis of ribosomal features involved in dynamic aspects of protein biosynthesis that are partially or fully disordered in H50S revealed the conformations of intersubunit bridges in unbound subunits, suggesting how they may change upon subunit association and how movements of the L1-stalk may facilitate the exit of tRNA.

Structural basis for the interaction of antibiotics with the peptidyl transferase centre in eubacteria

Ribosomes, the site of protein synthesis, are a major target for natural and synthetic antibiotics. Detailed knowledge of antibiotic binding sites is central to understanding the mechanisms of drug action. Conversely, drugs are excellent tools for studying the ribosome function. To elucidate the structural basis of ribosome–antibiotic interactions, we determined the high-resolution X-ray structures of the 50S ribosomal subunit of the eubacterium Deinococcus radiodurans, complexed with the clinically relevant antibiotics chloramphenicol, clindamycin and the three macrolides erythromycin, clarithromycin and roxithromycin. We found that antibiotic binding sites are composed exclusively of segments of 23S ribosomal RNA at the peptidyl transferase cavity and do not involve any interaction of the drugs with ribosomal proteins. Here we report the details of antibiotic interactions with the components of their binding sites. Our results also show the importance of putative Mg+2 ions for the binding of some drugs. This structural analysis should facilitate rational drug design.

Crystal structures of complexes of the small ribosomal subunit with tetracycline, edeine and IF3.

The small ribosomal subunit is responsible for the decoding of genetic information and plays a key role in the initiation of protein synthesis. We analyzed by X‐ray crystallography the structures of three different complexes of the small ribosomal subunit of Thermus thermophilus with the A‐site inhibitor tetracycline, the universal initiation inhibitor edeine and the C‐terminal domain of the translation initiation factor IF3. The crystal structure analysis of the complex with tetracycline revealed the functionally important site responsible for the blockage of the A‐site. Five additional tetracycline sites resolve most of the controversial biochemical data on the location of tetracycline. The interaction of edeine with the small subunit indicates its role in inhibiting initiation and shows its involvement with P‐site tRNA. The location of the C‐terminal domain of IF3, at the solvent side of the platform, sheds light on the formation of the initiation complex, and implies that the anti‐association activity of IF3 is due to its influence on the conformational dynamics of the small ribosomal subunit.

Structural insight into the role of the ribosomal tunnel in cellular regulation.

Nascent proteins emerge out of ribosomes through an exit tunnel, which was assumed to be a firmly built passive path. Recent biochemical results, however, indicate that the tunnel plays an active role in sequence-specific gating of nascent chains and in responding to cellular signals. Consistently, modulation of the tunnel shape, caused by the binding of the semi-synthetic macrolide troleandomycin to the large ribosomal subunit from Deinococcus radiodurans, was revealed crystallographically. The results provide insights into the tunnel dynamics at high resolution. Here we show that, in addition to the typical steric blockage of the ribosomal tunnel by macrolides, troleandomycin induces a conformational rearrangement in a wall constituent, protein L22, flipping the tip of its highly conserved β-hairpin across the tunnel. On the basis of mutations that alleviate elongation arrest, the tunnel motion could be correlated with sequence discrimination and gating, suggesting that specific arrest motifs within nascent chain sequences may induce a similar gating mechanism.

Induced-fit tightens pleuromutilins binding to ribosomes and remote interactions enable their selectivity

New insights into functional flexibility at the peptidyl transferase center (PTC) and its vicinity were obtained by analysis of pleuromutilins binding modes to the ribosome. The crystal structures of Deinococcus radiodurans large ribosomal subunit complexed with each of three pleuromutilin derivatives: retapamulin (SB-275833), SB-280080, and SB-571519, show that all bind to the PTC with their core oriented similarly at the A-site and their C14 extensions pointing toward the P-site. Except for an H-bond network with a single nucleotide, G2061, which involves the essential keto group of all three compounds, only minor hydrophobic contacts are formed between the pleuromutilin C14 extensions and any ribosomal component, consistent with the PTC tolerance to amino acid diversity. Efficient drug binding mode is attained by a mechanism based on induced-fit motions exploiting the ribosomal intrinsic functional flexibility and resulting in conformational rearrangements that seal the pleuromutilin-binding pocket and tightens it up. Comparative studies identified a network of remote interactions around the PTC, indicating that pleuromutilins selectivity is acquired by nonconserved nucleotides residing in the PTC vicinity, in a fashion resembling allosterism. Likewise, pleuromutilin resistant mechanisms involve nucleotides residing in the environs of the binding pocket, consistent with their slow resistance-development rates.

Symmetry at the active site of the ribosome: structural and functional implications

Abstract The sizable symmetrical region, comprising 180 ribosomal RNA nucleotides, which has been identified in and around the peptidyl transferase center (PTC) in crystal structures of eubacterial and archaeal large ribosomal subunits, indicates its universality, confirms that the ribosome is a ribozyme and evokes the suggestion that the PTC evolved by gene fusion. The symmetrical region can act as a center that coordinates amino acid polymerization by transferring intra-ribosomal signals between remote functional locations, as it connects, directly or through its extensions, the PTC, the three tRNA sites, the tunnel entrance, and the regions hosting elongation factors. Significant deviations from the overall symmetry stabilize the entire region and can be correlated with the shaping and guiding of the motion of the tRNA 3′-end from the A- into the P-site. The linkage between the elaborate PTC architecture and the spatial arrangements of the tRNA 3′-ends revealed the rotatory mechanism that integrates peptide bond formation, translocation within the PTC and nascent protein entrance into the exit tunnel. The positional catalysis exerted by the ribosome places the reactants in stereochemistry close to the intermediate state and facilitates the catalytic contribution of the P-site tRNA 2′-hydroxyl.

Crystallographic Studies on the Ribosome, a Large Macromolecular Assembly Exhibiting Severe Nonisomorphism, Extreme Beam Sensitivity and No Internal Symmetry

Crystals, diffracting best to around 3 A, have been grown from intact large and small ribosomal subunits. The bright synchrotron radiation necessary for the collection of the higher-resolution X-ray diffraction data introduces significant decay even at cryo temperatures. Nevertheless, owing to the reasonable isomorphism of the recently improved crystals of the small ribosomal subunits, reliable phases have been extracted at medium resolution (5-6 A) and an interpretable five-derivative MIR map has been constructed. For the crystals of the large subunits, however, the situation is more complicated because at higher resolution (2.7-7 A) they suffer from substantial radiation sensitivity, a low level of isomorphism, instability of the longest unit-cell axis and nonisotropic mosaicity. The 8 A MIR map, constructed to gain insight into this unusual system, may provide feasible reasoning for the odd combination of the properties of these crystals as well as hints for future improvement. Parallel efforts, in which electron-microscopy-reconstructed images are being exploited for molecular-replacement studies, are also discussed.

The transition state for formation of the peptide bond in the ribosome

Using quantum mechanics and exploiting known crystallographic coordinates of tRNA substrate located in the ribosome peptidyl transferase center around the 2-fold axis, we have investigated the mechanism for peptide-bond formation. The calculation is based on a choice of 50 atoms assumed to be important in the mechanism. We used density functional theory to optimize the geometry and energy of the transition state (TS) for peptide-bond formation. The TS is formed simultaneously with the rotatory motion enabling the translocation of the A-site tRNA 3′ end into the P site, and we estimated the magnitude of rotation angle between the A-site starting position and the place at which the TS occurs. The calculated TS activation energy, Ea, is 35.5 kcal (1 kcal = 4.18 kJ)/mol, and the increase in hydrogen bonding between the rotating A-site tRNA and ribosome nucleotides as the TS forms appears to stabilize it to a value qualitatively estimated to be ≈18 kcal/mol. The optimized geometry corresponds to a structure in which the peptide bond is being formed as other bonds are being broken, in such a manner as to release the P-site tRNA so that it may exit as a free molecule and be replaced by the translocating A-site tRNA. At TS formation the 2′ OH group of the P-site tRNA A76 forms a hydrogen bond with the oxygen atom of the carboxyl group of the amino acid attached to the A-site tRNA, which may be indicative of its catalytic role, consistent with recent biochemical experiments.

The evolving ribosome: from non-coded peptide bond formation to sophisticated translation machinery

Structural analysis supported by biochemical, mutagenesis and computational evidence, revealed that the contemporary ribosomes active site is a universal symmetrical pocket made of ribosomal RNA. This pocket seems to be the remnant of the proto-ribosome, a dimeric RNA assembly evolved by gene duplication, capable of autonomously catalyzing peptide bond formation and non-coded amino acid polymerization.