Interplay between the ribosomal tunnel, nascent chain, and macrolides influences drug inhibition.

Accumulating evidence suggests that, during translation, nascent chains can form specific interactions with ribosomal exit tunnel to regulate translation and promote initial folding events. The clinically important macrolide antibiotics bind within the exit tunnel and inhibit translation by preventing progression of the nascent chain and inducing peptidyl-tRNA drop-off. Here, we have synthesized amino acid- and peptide-containing macrolides, which are used to demonstrate that distinct amino acids and peptides can establish interaction with components of the ribosomal tunnel and enhance the ribosome-binding and inhibitory properties of the macrolide drugs, consistent with the concept that the exit tunnel is not simply a Teflon-like channel.

The oxazolidinone antibiotics perturb the ribosomal peptidyl-transferase center and effect tRNA positioning.

The oxazolidinones represent the first new class of antibiotics to enter into clinical usage within the past 30 years, but their binding site and mechanism of action has not been fully characterized. We have determined the crystal structure of the oxazolidinone linezolid bound to the Deinococcus radiodurans 50S ribosomal subunit. Linezolid binds in the A site pocket at the peptidyltransferase center of the ribosome overlapping the aminoacyl moiety of an A-site bound tRNA as well as many clinically important antibiotics.

Translational regulation via L11: molecular switches on the ribosome turned on and off by thiostrepton and micrococcin.

The thiopeptide class of antibiotics targets the GTPase-associated center (GAC) of the ribosome to inhibit translation factor function. Using X-ray crystallography, we have determined the binding sites of thiostrepton (Thio), nosiheptide (Nosi), and micrococcin (Micro), on the Deinococcus radiodurans large ribosomal subunit. The thiopeptides, by binding within a cleft located between the ribosomal protein L11 and helices 43 and 44 of the 23S rRNA, overlap with the position of domain V of EF-G, thus explaining how this class of drugs perturbs translation factor binding to the ribosome.

Cryo-EM study of the spinach chloroplast ribosome reveals the structural and functional roles of plastid-specific ribosomal proteins.

Protein synthesis in the chloroplast is carried out by chloroplast ribosomes (chloro-ribosome) and regulated in a light-dependent manner. Chloroplast or plastid ribosomal proteins (PRPs) generally are larger than their bacterial counterparts, and chloro-ribosomes contain additional plastid-specific ribosomal proteins (PSRPs); however, it is unclear to what extent these proteins play structural or regulatory roles during translation. We have obtained a three-dimensional cryo-EM map of the spinach 70S chloro-ribosome, revealing the overall structural organization to be similar to bacterial ribosomes.

A snapshot of the 30S ribosomal subunit capturing mRNA via the Shine-Dalgarno interaction.

In the initiation phase of bacterial translation, the 30S ribosomal subunit captures mRNA in preparation for binding with initiator tRNA. The purine-rich Shine-Dalgarno (SD) sequence, in the 5' untranslated region of the mRNA, anchors the 30S subunit near the start codon, via base pairing with an anti-SD (aSD) sequence at the 3' terminus of 16S rRNA. Here, we present the 3.

The antibiotic kasugamycin mimics mRNA nucleotides to destabilize tRNA binding and inhibit canonical translation initiation.

Kasugamycin (Ksg) specifically inhibits translation initiation of canonical but not of leaderless messenger RNAs. Ksg inhibition is thought to occur by direct competition with initiator transfer RNA. The 3.

X-ray crystallography study on ribosome recycling: the mechanism of binding and action of RRF on the 50S ribosomal subunit.

This study presents the crystal structure of domain I of the Escherichia coli ribosome recycling factor (RRF) bound to the Deinococcus radiodurans 50S subunit. The orientation of RRF is consistent with the position determined on a 70S-RRF complex by cryoelectron microscopy (cryo-EM). Alignment, however, requires a rotation of 7 degrees and a shift of the cryo-EM RRF by a complete turn of an alpha-helix, redefining the contacts established with ribosomal components.

Ribosomal crystallography: a flexible nucleotide anchoring tRNA translocation, facilitates peptide-bond formation, chirality discrimination and antibiotics synergism.

The linkage between internal ribosomal symmetry and transfer RNA (tRNA) positioning confirmed positional catalysis of amino-acid polymerization. Peptide bonds are formed concurrently with tRNA-3' end rotatory motion, in conjunction with the overall messenger RNA (mRNA)/tRNA translocation. Accurate substrate alignment, mandatory for the processivity of protein biosynthesis, is governed by remote interactions.

Ribosomal crystallography: peptide bond formation and its inhibition.

Ribosomes, the universal cellular organelles catalyzing the translation of genetic code into proteins, are protein/RNA assemblies, of a molecular weight 2.5 mega Daltons or higher. They are built of two subunits that associate for performing protein biosynthesis.

Structural insight into the antibiotic action of telithromycin against resistant mutants.

The crystal structure of the ketolide telithromycin bound to the Deinococcus radiodurans large ribosomal subunit shows that telithromycin blocks the ribosomal exit tunnel and interacts with domains II and V of the 23S RNA. Comparisons to other clinically relevant macrolides provided structural insights into its enhanced activity against macrolide-resistant strains.

On peptide bond formation, translocation, nascent protein progression and the regulatory properties of ribosomes. Derived on 20 October 2002 at the 28th FEBS Meeting in Istanbul.

High-resolution crystal structures of large ribosomal subunits from Deinococcus radiodurans complexed with tRNA-mimics indicate that precise substrate positioning, mandatory for efficient protein biosynthesis with no further conformational rearrangements, is governed by remote interactions of the tRNA helical features. Based on the peptidyl transferase center (PTC) architecture, on the placement of tRNA mimics, and on the existence of a two-fold related region consisting of about 180 nucleotides of the 23S RNA, we proposed a unified mechanism integrating peptide bond formation, A-to-P site translocation, and the entrance of the nascent protein into its exit tunnel. This mechanism implies sovereign, albeit correlated, motions of the tRNA termini and includes a spiral rotation of the A-site tRNA-3' end around a local two-fold rotation axis, identified within the PTC.

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.

Structural basis of the ribosomal machinery for peptide bond formation, translocation, and nascent chain progression.

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.

Antibiotics targeting ribosomes: crystallographic studies.

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.

On the interaction of colicin E3 with the ribosome.

Colicin E3 is a protein that kills Escherichia coli cells by a process that involves binding to a surface receptor, entering the cell and inactivating its protein biosynthetic machinery. Colicin E3 kills cells by a catalytic mechanism of a specific ribonucleolytic cleavage in 16S rRNA at the ribosomal decoding A-site between A1493 and G1494 (E. coli numbering system).

Initiation and inhibition of protein biosynthesis studies at high resolution.

Analysis of the high resolution structure of the small subunit from Thermus thermophilus shed light on its inherent conformational variability and indicated an interconnected network of features allowing concerted movements during translocation. It also showed that conformational rearrangements may be involved in subunit association and that a latch-like movement guarantees processivity and ensures maximized fidelity. Conformational mobility is associated with the binding and the anti association function of initiation factor 3, and antibiotics interfering with prevent the initiation of the biosynthetic process.

Ribosomal crystallography: from poorly diffracting microcrystals to high-resolution structures.

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.

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.

Structure of functionally activated small ribosomal subunit at 3.3 angstroms 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.

Targeting exposed RNA regions in crystals of the small ribosomal subunits at medium resolution.

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.