Structure Of Large Ribosomal Subunit Research Articles

Mutations Outside the Anisomycin-Binding Site Can Make Ribosomes Drug-Resistant

Eleven mutations that make Haloarcula marismortui resistant to anisomycin, an antibiotic that competes with the amino acid side chains of aminoacyl tRNAs for binding to the A-site cleft of the large ribosomal unit, have been identified in 23S rRNA. The correlation observed between the sensitivity of H. marismortui to anisomycin and the affinity of its large ribosomal subunits for the drug indicates that its response to anisomycin is determined primarily by the binding of the drug to its large ribosomal subunit. The structures of large ribosomal subunits containing resistance mutations show that these mutations can be divided into two classes: (1) those that interfere with specific drug–ribosome interactions and (2) those that stabilize the apo conformation of the A-site cleft of the ribosome relative to its drug-bound conformation. The conformational effects of some mutations of the second kind propagate through the ribosome for considerable distances and are reversed when A-site substrates bind to the ribosome.

Chemical parameters influencing fine-tuning in the binding of macrolide antibiotics to the ribosomal tunnel

Abstract In comparison to existing structural, biochemical, and therapeutical data, the crystal structures of large ribosomal subunit from the eubacterial pathogen model Deinococcus radiodurans in complex with the 14-membered macrolides erythromycylamine, RU69874, and the 16-membered macrolide josamycin, highlighted the similarities and differences in macrolides binding to the ribosomal tunnel. The three compounds occupy the macrolide binding pocket with their desosamine or mycaminose aminosugar, the C4-C7 edge of the macrolactone ring and the cladinose sugar sharing similar positions and orientations, although the latter, known to be unnecessary for antibiotic activity, displays fewer contacts. The macrolactone ring displays altogether few contacts with the ribosome and can, therefore, tilt in order to optimize its interaction with the 23S rRNA. In addition to their contacts with nucleotides of domain V of the 23S RNA, erythromycylamine and RU69874 interact with domain II nucleotide U790, and RU69874 also reaches van der Waals distance from A752, in a fashion similar to that observed for the ketolides telithromycin and cethromycin. The variability in the sequences and consequently the diversity of the conformations of macrolide binding pockets in various bacterial species can explain the drug's altered level of effectiveness on different organisms and is thus an important factor in structure-based drug design.

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. PTC features, ensuring the precise orientation required for the A-site nucleophilic attack on the P-site carbonyl-carbon, guide these motions. Solvent mediated hydrogen transfer appears to facilitate peptide bond formation in conjunction with the spiral rotation. The detection of similar two-fold symmetry-related regions in all known structures of the large ribosomal subunit, indicate the universality of this mechanism, and emphasizes the significance of the ribosomal template for the precise alignment of the substrates as well as for accurate and efficient translocation. The symmetry-related region may also be involved in regulatory tasks, such as signal transmission between the ribosomal features facilitating the entrance and the release of the tRNA molecules. The protein exit tunnel is an additional feature that has a role in cellular regulation. We showed by crystallographic methods that this tunnel is capable of undergoing conformational oscillations and correlated the tunnel mobility with sequence discrimination, gating and intracellular regulation.

5 S rRNA: structure and interactions.

5 S rRNA is an integral component of the large ribosomal subunit in all known organisms. Despite many years of intensive study, the function of 5 S rRNA in the ribosome remains unknown. Advances in the analysis of ribosome structure that have revealed the crystal structures of large ribosomal subunits and of the complete ribosome from various organisms put the results of studies on 5 S rRNA in a new perspective. This paper summarizes recently published data on the structure and function of 5 S rRNA and its interactions in complexes with proteins, within and outside the ribosome.

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. 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.

High-resolution structures of large ribosomal subunits from mesophilic eubacteria and halophilic archaea at various functional States.

Structural analysis of the recently determined high resolution structures of the small and the large ribosomal subunits from three bacterial sources, assisted by the medium resolution structure of a complex of the entire ribosome with three tRNAs, led to a quantum jump in our understanding of the process of the translation of the genetic code into proteins. Results of these studies highlighted dynamic aspects of protein biosynthesis; illuminated the modes of action of several antibiotics; indicated strategies adopted by ribosomes for maximizing their functional activity and revealed a wealth of architectural elements, including long tails of proteins penetrating the particle s cores and stabilizing the intricate folds of the RNA chains. Binding of substrate analogues showed that the decoding and the peptide-bond formation are accomplished mainly by RNA. However, several proteins may be functionally relevant in directing the mRNA and in mediating the proper orientation of the tRNA molecules within the ribosomal rRNA frame. Elements involved in intersubunit contacts or in substrate binding are inherently flexible, but maintain well-ordered characteristic conformations in unbound particles. The ribosomes utilize this conformational variability for optimizing their efficiency and minimizing non-productive interactions, hence disorder of functionally relevant features may be linked to less active conformations or to far from physiological conditions. Clinically relevant antibiotics bind almost exclusively to rRNA. In the small subunit they affect the decoding accuracy or limit conformational mobility and in the large subunit they either interfere with substrate binding, by interacting with components of the peptidyl transferase cavity, or hinder the progression of the growing peptide chain.