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New and improved antibiotics are urgently needed to combat the ever-increasing number of multidrug-resistant bacteria. In this study, we characterized several members of a new oxazolidinone family, Rχ-01. This antibiotic family is distinguished by having in vitro and in vivo activity against hospital-acquired, as well as community-acquired, pathogens. We compared the 50S ribosome binding affinity of this family to that of the only marketed oxazolidinone antibiotic, linezolid, using chloramphenicol and puromycin competition binding assays. The competition assays demonstrated that several members of the Rχ-01 family displace, more effectively than linezolid, compounds known to bind to the ribosomal A site. We also monitored binding by assessing whether Rχ-01 compounds protect U2585 ( Escherichia coli numbering), a nucleotide that influences peptide bond formation and peptide release, from chemical modification by carbodiimide. The Rχ-01 oxazolidinones were able to inhibit translation of ribosomes isolated from linezolid-resistant Staphylococcus aureus at submicromolar concentrations. This improved binding corresponds to greater antibacterial activity against linezolid-resistant enterococci. Consistent with their ribosomal A-site targeting and greater potency, the Rχ-01 compounds promote nonsense suppression and frameshifting to a greater extent than linezolid. Importantly, the gain in potency does not impact prokaryotic specificity as, like linezolid, the members of the Rχ-01 family show translation 50% inhibitory concentrations that are at least 100-fold higher for eukaryotic than for prokaryotic ribosomes. This new family of oxazolidinones distinguishes itself from linezolid by having greater intrinsic activity against linezolid-resistant isolates and may therefore offer clinicians an alternative to overcome linezolid resistance. A member of the Rχ-01 family of compounds is currently undergoing clinical trials.

This chapter focuses on the electron density map of the small ribosomal subunit, shows features interpreted as ribosomal proteins and rRNA, and pinpoints secondary-structure elements. It highlights the use of heavy-atom markers for unbiased targeting of surface rRNA (e.g., the 3 end of the 16S RNA) and for the localization of proteins TS11 and TS13. Efforts to induce controlled conformational changes within the crystals are also discussed in the chapter. The globular regions of lower density could be assigned to folds observed in isolated ribosomal proteins as determined by nuclear magnetic resonance (NMR) and crystallography at atomic resolution. The globular regions seen in the maps, most of which are of lower average density, were found appropriate to accommodate ribosomal proteins. The main chain coordinates, as determined by X-ray crystallography or NMR for the isolated proteins at high resolution, were used as templates. The chapter talks about structural markers targeted to predetermined sites, and functional activation in pre-and postcrystallization states. Despite severe crystallographic problems, the way to structure determination has been paved and electron density maps at close to molecular resolution are emerging. The growing popularity of ribosomal crystallography is indeed gratifying. This, together with the fruitful interactions with the exciting advances in cryo-EM, is bound to lead to major breakthroughs.

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.

The cellular organelles translating the genetic code into proteins, the ribosomes, are large, asymmetric, flexible, and unstable ribonucleoprotein assemblies, hence they are difficult to crystallize. Despite two decades of intensive effort and thorough searches for suitable sources, so far only three crystal types have yielded high-resolution structures: two large subunits (from an archaean and from a mesophilic eubacterium) and one thermophilic small subunit. These structures have added to our understanding of decoding, have revealed dynamic aspects of the biosynthetic process, and have indicated the strategies adopted by ribosomes for interacting between themselves as well as with inhibitors, factors and substrates.

Crystals of the small ribosomal subunit from Thermus thermophilus diffract to 3A and exhibit reasonable isomorphism and moderate resistance to irradiation. A 5A MIR map of this particle shows a similar shape to the part assigned to this particle within the cryo-EM reconstructions of the whole ribosome and contains regions interpretable either as RNA chains or as protein motifs. To assist phasing at higher resolution we introduced recombinant methods aimed at extensive selenation for MAD phasing. We are focusing on several ribosomal proteins that can be quantitatively detached by chemical means. These proteins can be modified and subsequently reconstituted into depleted ribosomal cores. They also can be used for binding heavy atoms, by incorporating chemically reactive binding sites, such as -SH groups, into them. In parallel we are co-crystallizing the ribosomal particles with tailor made ligands, such as antibiotics or cDNA to which heavy-atoms have been attached or diffuse the latter compounds into already formed crystals.

Crystals of small and large ribosomal subunits from thermophilic and halophilic bacteria, diffracting to 3 A, are being subjected to structural analysis with synchrotron radiation. The bright beam necessary for detecting and collecting the diffraction at the higher-resolution shell causes significant decay even at 25 K. Nevertheless, data collected from native and heavy-atom-derivatized crystals led to the construction of electron density maps of both ribosomal subunits, showing recognizable morphologies and internal features similar to those observed by EM reconstructions of the corresponding ribosomal particle. The main features of these maps include elongated dense regions traceable as well separated RNA duplexes or single strands. Also seen are globular patches of lower density, readily distinguishable from the above, in which folds observed by NMR or crystallography in isolated ribosomal proteins at atomic resolution were detected. The intercomponents contacts identified so far reveal diverse modes of recognition. Metal clusters, attached at selected sites on the particles, are being exploited to facilitate unbiased map interpretation. In this way, two surface proteins were located and several surface RNA strands were targeted.

DbpA is a putative Escherichia coli ATP dependent RNA helicase belonging to the family of DEAD box proteins. It hydrolyzes ATP in the presence of 23S ribosomal RNA and 93 bases in the peptidyl transferase center of 23S rRNA are sufficient to trigger 100% of the ATPase activity of DbpA. In the present study we characterized the ATPase and RNA unwinding activities of DbpA in more detail. We report that-in contrast to eIF-4A, the prototype of the DEAD box protein family-the ATPase and the helicase activities of DbpA are not coupled. Moreover, the RNA unwinding activity of DbpA is not specific for 23S rRNA, since DbpA is also able to unwind 16S rRNA hybrids. Furthermore, we determined that the ATPase activity of DbpA is triggered to a significant extent not only by the 93 bases of the 23S rRNA previously reported but also by other regions of the 23S rRNA molecule. Since all these regions of 23S rRNA are either part of the 'functional core' of the 50S ribosomal subunit or involved in the 50S assembly, DbpA may play an important role in the ribosomal assembly process.

Preliminary phases were determined by the application of the isomorphous replacement method at low and intermediate resolution for structure factor amplitudes collected from crystals of large and small ribosomal subunits from halophilic and thermophilic bacteria. Derivatization was performed with dense heavy atom clusters, either by soaking or by specific covalent binding prior to the crystallization. The resulting initial electron density maps contain features comparable in size to those expected for the corresponding particles. The packing arrangements of these maps have been compared with motifs observed by electron microscopy in positively stained thin sections of embedded three-dimensional as well as with phase sets obtained by ab-initio computations. Aimed at higher resolution phasing, procedures are being developed for multi-site binding of relatively small dense metal clusters at selected locations. Potential sites are being inserted either by mutagenesis or by chemical modifications to facilitate cluster binding to the large halophilic and the small thermophil!c ribosomal subunits which yield crystals diffracting to the highest resolution obtained so far for ribosomes, 2.9 and 7.3 A, respectively. For this purpose the surfaces of these ribosomal particles have been characterized and conditions for quantitative reversible detachment of selected ribosomal proteins have been found. The corresponding genes are being cloned, sequenced, mutated to introduce the reactive side-groups (mainly cysteines) and overexpressed. To assist the interpretation of the anticipated electron density maps, sub-ribosomal stable complexes were isolated from H50S. One of these complexes is composed of two proteins and the other is made of a stretch of the rRNA and a protein. For exploiting the exposed parts of the surface of these complexes for heavy atom binding and for attempting the determination of their three-dimensional structure, their components are being produced genetically. The low resolution models reconstructed from tilt series of crystalline arrays of ribosomal particles are being employed for initial phasing. The tentative functional interpretation of these models stimulated the design and the crystallization of complexes mimicking

Preliminary electron density maps of the large and the small ribosomal particles from halophilic and thermophilic sources, phased by the isomorphous replacement method, have been constructed at intermediate resolution. These maps contain features comparable in size with what is expected for the corresponding particles, and their packing arrangements are in accord with the schemes obtained by ab-initio procedures as well as with the motifs observed in thin sections of the crystals by electron microscopy. To phase higher resolution data, procedures are being developed for derivatization by specific labeling of the ribosomal particles at selected locations with rather small and dense clusters. Potential binding sites are being inserted either by site directed mutagenesis or by chemical modifications to facilitate cluster binding on the surface of the halophilic large and the thermophilic small ribosomal particles, which yield the crystals diffracting to highest resolution (2.9 and 7.3 Å (1 Å = 0.1 nm), respectively). For this purpose, the surface of these ribosomal particles is being characterized and procedures are being developed for quantitative detachment of selected ribosomal proteins and for their incorporation into core particles. The genes of these proteins are being cloned, sequenced, mutated to introduce reactive side groups, mainly cysteines, and overexpressed. In parallel, two in situ small and stable complexes were isolated from the halophilic ribosome. Procedures for their crystal production in large quantities are currently being developed. Models, reconstructed at low resolution from crystalline arrays of ribosomes and their large subunits, are being used for initial low-resolution phasing of the X-ray amplitudes. The interpretation of these models stimulated the design and the crystallization of complexes mimicking defined functional states of a higher quality than those obtained for isolated ribosomes. These models also inspired modelling experiments according to results of functional studies, performed elsewhere, focusing on the progression of nascent proteins.Key words: ribosomes, crystallography, undecagold cluster, heteropolyanions.

Crystals of various ribosomal particles, diffracting best to 2.9 A resolution were grown. Crystallographic data were collected from shock frozen crystals with intense synchrotron radiation at cryo temperature. For obtaining phase information, monofunctional reagents were prepared from an undecagold and a tetrairidium cluster, by attaching to them chemically reactive handles, specific for sulfhydryl moieties. Heavy-atom derivatives were prepared by a specific and quantitative binding of the undecagold cluster to an exposed sulfhydryl prior to the crystallization. To create potential binding sites on the halophilic and thermophilic ribosomal particles, which yield our best and most interesting crystals, exposed reactive moieties were inserted, using genetic and chemical procedures. In order to choose the appropriate locations for these insertions, the surfaces of the ribosomal particles were mapped by direct chemical determination of exposed amino and sulfhydryl groups.

An electron density map of the large ribosomal subunit from Bacillus stearothermophilus was obtained at 26 A resolution by single isomorphous replacement (SIR) from a derivative formed by specific quantitative labeling with a dense undecagold cluster. For derivalization, a mono-functional reagent of this cluster was bound to a sulfhydryl group of a purified ribosomal protein. which was in turn reconstituted with core particles of a mutant lacking this protein. The native, mutated, and derivatzed 50S ribosomal subunits crystallize under the same conditions in the same space group. Under favorable conditions, crystals of the derivatized subunit proved to be isomorphous with the native ones, whereas the crystals of the mutant may have somewhat different packing. After resolving the SIR phase ambiguity by solvent flattening, the electron density shows a packing that is consistent with the noncrystallographic symmetry found by Patterson searches as well as with the motif observed in electron micrographs of thin sections of the crystals. These studies established that phase information can be obtained from heavy metal clusters, even when the crystals under investigation are unstable and weakly diffracting. These results encouraged further effort at the construction of specifically derivatized crystals from other ribosomal particles that diffract to higher resolution. © 1994 John Wiley & Sons, Inc.

Crystals of 5OS ribosomal subunits from Haloarcula marismortui diffracting to 2.9 A resolution were grown. Because of their large unit cells and the extremely weak diffracting power, almost all X-ray crystallographic analysis of these crystals must be performed with intense synchrotron radiation. At ambient temperature, all ribosomal crystals decay upon the first instance of X-irradiation. To overcome this severe sensitivity, procedures for data collection at cryo temperature were developed. Under these conditions the crystals can be irradiated for periods sufficient for the collection of more than one data set from an individual crystal (days or weeks) with no observable damage. They also can be stored for months, to resume interrupted measurements. To assist the interpretation of the anticipated electron density map, a specific internal nucleoprotein complex of protein HmaL1 and a stretch of H23S rRNA was isolated from the halophilic ribosome. The fragments of the 23S rRNA protected by the protein from nuclease digestion were sequenced. Alignment of the sequences of some archaebacterial L1-specific RNA fragments to the corresponding parts of eubacterial and eukaryotic rDNAs, localized the sequence identities to two distinct regions. Chimeric complexes were reconstituted with the corresponding E. coli ribosomal components, indicating a rather high homology, despite the evolution distance. A feasible secondary structure of the rRNA stretch participating in this complex was found to be compatible with the one proposed for the corresponding part in the E. coli ribosomal RNA.

Impressive progress has been made towards the determination of the molecular structure of the ribosome and the illumination of its functional features. The highlights include reaching almost atomic resolution in crystallography of intact ribosomal particles and isolated ribosomal components and the assignments of several functional centers.

Summary Ribosomal particles from Haloarcula marismortui were crystallized. The best crystals, diffracting to 2.9 A resolution and yielding crystallographic data of reasonable quality were grown from the large ribosomal subunits. Attempts at crystallization of functional ribosomal complexes are in progress, benefiting from experience gained from crystals of ribosomes of an extreme thermophilic bacterium, Thermus thermophilus . For obtaining phase information, a monofunctional reagent was prepared from an undecagold cluster, by attaching to it a chemically reactive handle, specific for sulfhydryl moieties. Heavy atom derivatives were prepared by binding this cluster to exposed sulfhydryls prior to the crystallization. Cores of halophilic ribosomal particles, lacking four ribosomal proteins, were prepared using dioxane. All detached proteins could be fully reconstituted. However, blocking the -SH group of one of them (HmaL11), prevented its incorporation into the core particles. The so obtained depleted 50S subunits crystallize under the same conditions as native ones and show apparent isomorphism with them. Most of the genes of several r-proteins were cloned. These are being used for sequencing as well as for providing new locations for binding heavy atom clusters by genetic insertion of cysteines on the ribosomal surface, according to accessibility data, obtained either chemically or by limited proteolysis. A nucleoprotein complex of protein HmaL1 and a fragment of the 23S rRNA was isolated from ribosomes. Chimeric complexes were reconstituted with E. coli ribosomal components, indicating rather high homolgy, despite the evolution distance.

A new, inherently flexible, predominantly α-helical RNA binding motif has been detected in two rather conserved ribosomal proteins. Comparative studies prompt the suggestion that these ribosomal proteins are the ancestors of this new helix-turn-helix fold, also found in homeodomains.

The high-resolution structures of the bacterial ribosomal subunits and those of their complexes with antibiotics have advanced significantly our understanding of small-molecule interactions with RNA. The wealth of RNA structural data generated by these structures has allowed computational chemists to employ a drug discovery paradigm focused on RNA-based targets. The structures also show how target-based resistance affects antibiotics acting at the level of the ribosome. Not only are the sites pinpointed where different classes of antibiotics inhibit protein synthesis, but their orientations, relative dispositions, and unique mechanisms of action are also revealed at the atomic level. Both the 30S and the 50S ribosomal subunits have been shown to be "targets of targets", offering several adjacent, functionally relevant binding pockets for antibiotics. It is the detailed knowledge of these validated locations, or ribofunctional loci, plus the mapping of the resistance hot-spots that allow the rational design of next-generation antibacterials. When the structural information is combined with a data-driven computational toolkit able to describe and predict molecular properties appropriate for bacterial cell penetration and drug-likeness, a structure-based drug design approach for novel antibacterials shows great promise.

Crystal structures of tRNA mimics complexed with the large ribosomal subunit of Deinococcus radiodurans indicate that remote interactions determine the precise orientation of tRNA in the peptidyl-transferase center (PTC). The PTC tolerates various orientations of puromycin derivatives and its flexibility allows the conformational rearrangements required for peptide-bond formation. Sparsomycin binds to A2602 and alters the PTC conformation. H69, the intersubunit-bridge connecting the PTC and decoding site, may also participate in tRNA placement and translocation. A spiral rotation of the 3′ end of the A-site tRNA around a 2-fold axis of symmetry identified within the PTC suggests a unified ribosomal machinery for peptide-bond formation, A-to-P-site translocation, and entrance of nascent proteins into the exit tunnel. Similar 2-fold related regions, detected in all known structures of large ribosomal subunits, indicate the universality of this mechanism.

Resistance to antibiotics is a major problem in modern therapeutics. Ribosomes, the cellular organelle catalyzing the translation of the genetic code into proteins, are targets for several clinically relevant antibiotics. The ribosomes from eubacteria are excellent pathogen models. High resolution structures of the large and small ribosomal subunits were used as references that allowed unambiguous localization of almost a dozen antibiotic drugs, most of which are clinically relevant. Analyses of these structures showed a great diversity in the antibiotics' modes of action, such as interference with substrate binding, hindrance of the mobility required for the biosynthetic process and the blockage of tunnel which provides the path of exit for nascent proteins. All antibiotics studied by us were found to bind primarily to ribosomal RNA and, except for one allosteric effect, their binding did not cause major conformational changes. Antibiotics of the small ribosomal subunit may hinder tRNA binding, decoding, translocation, and the initiation of the entire biosynthetic process. The large subunit agents may target the GTPase center, interfere with peptide bond formation, or block the entrance of the nascent protein exit tunnel. The overall structure of the peptidyl transferase center and the modes of action of the antibiotic agents indicate that the ribosome serves as a template for proper positioning of tRNAs, rather than participating actively in the catalytic events associated with the creation of peptide bonds.

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.

Ribosomes are complexes of ribosomal RNAs and proteins. As more primary structures of ribosomal proteins have been compiled amino acid motifs associated with nucleic acid binding have been identified in ribosomal proteins (Wool et al. 1995). Furthermore, the cloning and overexpression of ribosomal proteins has resulted in the elucidation of the motifs or domains implicated in rRNA binding (Ramakrishnan et al. 1995). For example the Bacillus stearothermophilus L11 was recently proven to bind rRNA through a helix-turn-helix domain identical to that of the “homeodomain” regulatory proteins (Xing et al. 1997).

50 S ribosomal subunits from Bacillus stearothermophilus have been crystallized as 2-dimensional periodic arrays on phospholipid monolayer films at the water-air interface. These crystals were preserved in vitreous ice and imaged with 100 keV electrons under low dose and low temperature conditions. The unit cell parameters of the crystals are a = 371.3(+/- 3.8) A, b = 152.3(+/- 1.6) A, gamma = 96.3(+/- 1.0) degrees. Some of the image arrays of these crystals have twofold rotational symmetry with a phase residual of less than 25 degrees. The mean figure of merit of the merged structure factors from these image arrays out to 20 A resolution is higher than 0.87. The 2-dimensional projection map shows a level of detail not seen in previous structural studies of the 50 S ribosome subunit. Some of these features may be related to the current 3-dimensional model of the subunit. This analysis illustrates the potential of using the electron crystallographic approach for determining the 3-dimensional structure of the 50 S ribosomal subunit crystallized on a monolayer surface. In addition, the structural information retrieved by electron crystallography might be useful for phasing X-ray data towards an atomic resolution model of the ribosome.

The assembly of the 50S subunit from Escherichia coli ribosomes is initiated by two ribosomal proteins, L24 and L3. A mutant lacking the assembly-initiator protein L24 shows distinct phenotypic features (temperature sensitivity, growth rate reduced by a factor of 6 at permissive temperatures below 34 degrees C, underproduction of 50S subunits), which could be traced back to assembly effects caused by lack of L24 [Herold, M., Nowotny, V., Dabbs, E. R., & Nierhaus, K. H. (1986) Mol. Gen. Genet. 203, 281-287]. As expected, only one assembly protein was effective during in vitro assembly at nonpermissive temperatures, whereas surprisingly the restoration of active particle formation at permissive temperatures was paralleled by the reappearance of two initiator proteins. Here we analyze the initiation of assembly at permissive temperatures in the absence of L24. We demonstrate in a series of reconstitution experiments with purified proteins that the two initiator proteins are L20 and L3. Thus, L20 can replace L24 for the initiation of assembly at permissive temperatures.

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 particle's 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.