ABSTRACT
A ribosome is a ribozyme polymerizing amino acids, exploiting positional- and substrate-mediated chemical catalysis. We showed that peptide-bond formation is facilitated by the ribosomal architectural frame, provided by a sizable symmetry-related region in and around the peptidyl transferase centre, suggesting that the ribosomal active site was evolved by gene fusion. Mobility in tunnel components is exploited for elongation arrest as well as for trafficking nascent proteins into the folding space bordered by the bacterial chaperone, namely the trigger factor.
Subject(s)
Evolution, Molecular , Peptide Chain Elongation, Translational , Protein Folding , Ribosomes/chemistry , Ribosomes/metabolism , Catalysis , Crystallography, X-Ray , Ribosomes/geneticsABSTRACT
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.
Subject(s)
Gram-Positive Cocci/chemistry , RNA, Ribosomal/chemistry , Ribosomal Proteins/chemistry , Ribosomes/chemistry , Bacterial Proteins/chemistry , Crystallography, X-Ray , Gram-Positive Cocci/ultrastructure , Macromolecular Substances , Models, Molecular , Molecular Structure , Nucleic Acid Conformation , Protein Conformation , Protein Structure, Secondary , Protein Structure, Tertiary , RNA, Bacterial/chemistry , RNA, Bacterial/metabolism , RNA, Transfer/chemistry , RNA, Transfer/metabolism , Ribosomes/ultrastructureABSTRACT
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.
Subject(s)
Anti-Bacterial Agents/metabolism , Bacteria/metabolism , Peptidyl Transferases/metabolism , Ribosomes/metabolism , Base Sequence , Binding Sites , Chloramphenicol/metabolism , Crystallography, X-Ray , Macrolides/metabolism , Magnesium/metabolism , Models, Molecular , Molecular Sequence Data , Protein Conformation , RNA, Bacterial/metabolism , RNA, Ribosomal, 23S/metabolism , Ribosomal Proteins/metabolism , Structure-Activity RelationshipABSTRACT
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.
Subject(s)
Edeine/chemistry , Peptide Chain Initiation, Translational , Peptide Initiation Factors/chemistry , Ribosomes/chemistry , Tetracycline/chemistry , Thermus thermophilus , Binding Sites , Crystallography, X-Ray , Eukaryotic Initiation Factor-3 , Models, Molecular , Protein Synthesis Inhibitors/chemistryABSTRACT
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.
Subject(s)
Crystallography, X-Ray/methods , Ribosomes/chemistry , Ribosomes/ultrastructure , Archaea/chemistry , Bacterial Proteins/chemistry , Microscopy, Electron , Models, Molecular , Protein Binding , Protein Conformation , Protein Structure, Tertiary , RNA/chemistry , X-Ray DiffractionABSTRACT
Within the framework of ribosomal crystallography, the small subunits are being analyzed, using crystals diffracting to 3 A resolution. The medium resolution electron density map of this subunit, obtained by multiple isomorphous replacement, show recognizable morphologies, strikingly similar to the functional active conformer of the small ribosomal subunit. It contains elongated dense features, traceable as RNA chains as well as globular regions into which the structures determined for isolated ribosomal proteins, or other known structural motifs were fitted. To facilitate unbiased map interpretation, metal clusters are being covalently attached either to the surface of the subunits or to DNA oligomers complementary to exposed ribosomal RNA. Two surface cysteines and the 3' end of the 16S ribosomal RNA have been localized. Targeting several additional RNA regions shed light on their relative exposure and confirmed previous studies concerning their functional relevance.
Subject(s)
RNA, Ribosomal/chemistry , Ribosomes/chemistry , Crystallography, X-Ray , Cysteine/chemistry , DNA, Complementary/chemistry , Macromolecular Substances , Models, Molecular , Nucleic Acid Conformation , Protein Conformation , RNA, Bacterial/chemistry , RNA, Ribosomal, 16S/chemistry , Ribosomal Proteins/chemistry , Static Electricity , Thermus thermophilus/chemistryABSTRACT
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.
Subject(s)
Nucleic Acid Conformation , RNA, Bacterial/chemistry , RNA, Bacterial/metabolism , Ribosomes/chemistry , Ribosomes/metabolism , Thermus thermophilus/chemistry , Base Pairing , Binding Sites , Crystallography, X-Ray , Models, Molecular , Protein Conformation , RNA, Bacterial/genetics , RNA, Messenger/chemistry , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Ribosomal, 16S/chemistry , RNA, Ribosomal, 16S/genetics , RNA, Ribosomal, 16S/metabolism , RNA, Transfer/chemistry , RNA, Transfer/genetics , RNA, Transfer/metabolism , Ribosomes/genetics , Structure-Activity Relationship , Thermus thermophilus/cytology , Thermus thermophilus/geneticsABSTRACT
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.
Subject(s)
Bacterial Proteins/chemistry , Ribosomal Proteins/chemistry , Thermus thermophilus/chemistry , Thermus thermophilus/genetics , Bacterial Proteins/isolation & purification , Binding Sites , Cloning, Molecular , Crystallography, X-Ray , DNA, Complementary/metabolism , Models, Molecular , Nucleic Acid Hybridization , Ribosomal Proteins/isolation & purificationABSTRACT
The electron density map of the small ribosomal subunit from Thermus thermophilus, constructed at 4.5 A resolution, shows the recognizable morphology of this particle, as well as structural features that were interpreted as ribosomal RNA and proteins. Unbiased assignments, carried out by quantitative covalent binding of heavy atom compounds at predetermined sites, led to the localization of the surface of the ribosomal protein S13 at a position compatible with previous assignments, whereas the surface of S11 was localized at a distance of about twice its diameter from the site suggested for its center by neutron scattering. Proteins S5 and S7, whose structures have been determined crystallographically, were visually placed in the map with no alterations in their conformations. Regions suitable to host the fold of protein S15 were detected in several positions, all at a significant distance from the location of this protein in the neutron scattering map. Targeting the 16S RNA region, where mRNA docks to allow the formation of the initiation complex by a mercurated mRNA analog, led to the characterization of its vicinity.
Subject(s)
Ribosomes/chemistry , Thermus thermophilus/chemistry , Base Sequence , Molecular Sequence Data , Protein Conformation , RNA, Messenger/metabolism , RNA, Ribosomal, 16S/metabolism , Ribosomal Proteins/chemistryABSTRACT
Procedures were developed exploiting organometallic clusters and coordination compounds in combination with heavy metal salts for derivatization of ribosomal crystals. These enabled the construction of multiple isomorphous replacement (MIR) and multiple isomorphous replacement combined with anomalous scattering medium-resolution electron density maps for the ribosomal particles that yield the crystals diffracting to the highest resolution, 3 A, of the large subunit from Haloarcula marismortui and the small subunit from Thermus thermophilus. The first steps in the interpretation of the 7. 3-A MIR map of the small subunit were made with the aid of a tetrairidium cluster that was covalently attached to exposed sulfhydryls on the particle's surface prior to crystallization. The positions of these sulfhydryls were localized in difference Fourier maps that were constructed with the MIR phases.
Subject(s)
Microscopy, Electron/methods , Organometallic Compounds/chemistry , Ribosomes/chemistry , Ribosomes/ultrastructure , Animals , Crystallography/methods , Image Processing, Computer-Assisted , Metals, Heavy/chemistry , Molecular Conformation , RNA, Ribosomal/chemistry , RNA, Ribosomal/ultrastructure , Ribosomal Proteins/chemistry , Ribosomal Proteins/ultrastructureABSTRACT
BACKGROUND: Ribosomes are the universal cellular organelles that accomplish the translation of the genetic code into proteins. Electron cryo-microscopy (cryo-EM) has yielded fairly detailed three-dimensional reconstructions of ribosomes. These were used to assist in the determination of higher resolution structures by X-ray crystallography. RESULTS: Molecular replacement studies using cryo-EM reconstructions provided feasible packing schemes for crystals of ribosomes and their two subunits from Thermus thermophilus, and of the large subunits from Haloarcula marismortui. For the large subunits, these studies also confirmed the major heavy-atom sites obtained by single isomorphous replacement combined with anomalous diffraction (SIRAS) and by multiple isomorphous replacement combined with anomalous diffraction (MIRAS) at approximately 10 A. Although adequate starting phases could not be obtained for the small subunits, the crystals of which diffract to 3.0 A, cryo-EM reconstructions were indispensable for analyzing their 7.2 A multiple isomorphous replacement (MIR) map. This work indicated that the conformation of the crystallized small subunits resembles that seen within the 70S ribosomes. Subsequently, crystals of particles trapped in their functionally active state were grown. CONCLUSIONS: Single-particle cryo-EM can contribute to the progress of crystallography of non-symmetrical, large and flexible macromolecular assemblies. Besides confirming heavy-atom sites, obtained from flat or overcrowded difference Patterson maps, the cryo-EM reconstructions assisted in elucidating packing arrangements. They also provided tools for the identification of the conformation within the crystals and for the estimation of the level of inherent non-isomorphism.
Subject(s)
Ribosomal Proteins/chemistry , Ribosomes/chemistry , Crystallography, X-Ray , Escherichia coli/chemistry , Microscopy, Electron/methods , Models, Molecular , Protein ConformationABSTRACT
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.
Subject(s)
Ribosomes/chemistry , Ribosomes/ultrastructure , Animals , Crystallography, X-Ray , Humans , X-Ray DiffractionABSTRACT
The structure of the mammalian ribosome, reconstructed at 25 A resolution, has added a new dimension to our current knowledge, as it manifests the conservation and universality of the ribosome in respect to its primary tasks in protein biosynthesis. A combined approach to study of the ribosome, using X-ray crystallography and electron microscopy, may further improve our understanding of ribosome function in the future.
Subject(s)
Evolution, Molecular , Ribosomes/chemistry , Animals , Crystallography, X-Ray , Mammals , Microscopy, Electron , Models, Molecular , Protein BiosynthesisABSTRACT
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.
Subject(s)
Bacteria/ultrastructure , Crystallography, X-Ray/methods , Ribosomes/ultrastructure , DNA, Complementary , Escherichia coli/ultrastructure , Freezing , Geobacillus stearothermophilus/ultrastructure , Halobacteriaceae/ultrastructure , RNA, Ribosomal/ultrastructure , Thermus thermophilus/ultrastructureABSTRACT
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.
Subject(s)
Geobacillus stearothermophilus/chemistry , Ribosomes/chemistry , Crystallography , Image Processing, Computer-Assisted , Microscopy, Electron/methods , Ribosomes/ultrastructureABSTRACT
Crystals of 50S 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.
Subject(s)
Ribonucleoproteins/isolation & purification , Ribosomes/ultrastructure , Base Sequence , Crystallography, X-Ray , Macromolecular Substances , Molecular Sequence Data , Nucleic Acid Conformation , Ribonucleoproteins/chemistry , Ribonucleoproteins/ultrastructure , Ribosomes/chemistryABSTRACT
A ribosomal protein, showing no homology with other known prokaryotic ribosomal proteins, was isolated and characterized from the thermophilic eubacteria, Thermus thermophilus, T. aquaticus and T. flavus. This small (26 amino acids) and strongly basic (1 acidic and 13 basic residues) protein displayed the same primary structure from all three sources. Interestingly, it shows about 65% homology with a ribosomal protein from spinach chloroplasts (J. Schmidt, personal communication).