Ribosomal Crystallography: Initiation, Peptide Bond Formation, and Amino Acid Polymerization are Hampered by Antibiotics

Abstract

■ Abstract High-resolution structures of ribosomal complexes revealed that minute amounts of clinically relevant antibiotics hamper protein biosynthesis by limiting ribosomal mobility or perturbing its elaborate architecture, designed for navigating and controlling peptide bond formation and continuous amino acid polymerization. To accomplish this, the ribosome contributes positional rather than chemical catalysis, provides remote interactions governing accurate substrate alignment within the flexible peptidyl-transferase center (PTC) pocket, and ensures nascent-protein chirality through spatial limitations. Peptide bond formation is concurrent with aminoacylatedtRNA 3 end translocation and is performed by a rotatory motion around the axis of a sizable ribosomal symmetry-related region, which is located around the PTC in all known crystal structures. Guided by ribosomal-RNA scaffold along an exact pattern, the rotatory motion results in stereochemistry that is optimal for peptide bond formation and for nascent protein entrance into the exit tunnel, the main target of antibiotics targeting ribosomes. By connecting the PTC, the decoding center, and the tRNA entrance and exit regions, the symmetry-related region can transfer intraribosomal signals, guaranteeing smooth processivity of amino acid polymerization. CONTENTS INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . INTRODUCTIONRibosomes are the universal cellular riboprotein assemblies that catalyze amino acid polymerization into proteins. In this process, called protein biosynthesis, the ribosomes interact with the cellular environment, select the genetic frame to be translated, form the peptide bonds, and act as polymerases to ensure smooth and efficient elongation of the newly born protein chains. The ribosomes are composed of two riboprotein subunits of unequal size that associate upon the initiation of the process and dissociate at its termination. Protein biosynthesis is performed by a cooperative effort of both subunits, and the ribosome possesses features that facilitate transmission between its various functional sites, leading to efficient processivity of protein formation essential for cell vitality. The small ribosomal subunit facilitates the initiation of the translation process and is involved in selecting the frame to be translated, decoding the genetic message, and controlling the fidelity of codon-anticodon interactions. The large ribosomal subunit forms the peptide bond, ensures smooth amino acid polymerization, and channels the nascent proteins through their exit tunnel. Messenger RNA (mRNA) carries the genetic instructions to the ribosome, and aminoacylated transfer RNA (tRNA) molecules deliver the amino acids within a ternary complex containing GTP and the elongation factor Tu. The ribosome possesses three tRNA binding sites. The A-site hosts the aminoacylated-tRNA, the P-site hosts the peptidyl tRNA, and the E-site designates the location of the exiting free tRNA once a p...

The pseudouridine synthase RluD is required for normal ribosome assembly and function in Escherichia coli

“…This region has been shown to be part of the peptidyltransferase center (PTC). Based on the crystal structures of Thermus thermophilus 70S ribosomes (Yusupov et al 2001) and Deinococcus radiodurans 50S rRNA (Bashan et al 2003;Yonath and Bashan 2004) and cryo-electron microscopy mapping of E. coli 70S ribosomes (Agrawal et al 2004) complexed with various substrates, domain IV and helix 69 (including C1915; Agrawal et al 2004) are known to interact with mRNA, tRNAs, 16S rRNA, and Ribosomal Release Factor, and may be involved in proper tRNA positioning, in translocation, and in release of mRNA from the posttermination complex. Taken together, these findings suggest that helix 69 and the modified residues of 23S rRNA are important for efficient protein biosynthesis by the ribosome.…”

Ribosome's mode of function: myths, facts and recent results

“…Indeed, this modification facilitated the binding of the drug also to the ribosomes from the archaeon H. marismortui [76], albeit without inhibitory action, since its mode of binding is different from that observed for eubacteria [81]. Thus, azithromycin binding to H50S blocks only a small part of the tunnel, whereas in D50S the binding yields an effective blockage of the exit tunnel [70,80].…”

“…The first widely used macrolide drug was erythromycin, a 14-member lactone ring, derivatized with a desosamine and cladinose sugar ( Figure 4). All currently available crystal structures of complexes of macrolides and their advanced derivatives with large ribosomal subunits [40,59,67,[76][77][78][79][80][81][82] show that most of the interactions of macrolides with the ribosome exit tunnel involve the main constituents of the macrolide-binding pocket, nucleotides A2058-A2059, which reside on one side of the tunnel wall, and provide the major elements allowing for drug selectivity (see below) and drug resistance. Erythromycin resistance can be acquired also by mutations in protein L22 tip, as well as in protein L4 that forms, together with L22, a constricted region of the tunnel, and can meet it in its swung conformation ( Figure 4).…”

“…Additionally, by revealing the structural factors that discriminate between the ribosomes from eubacteria and those of an archaeon representing eukaryotes, these structures illuminated basic issues in antibiotics susceptibility, specificity, selectivity, and toxicity [1,75,80,89]. As an example, the key nucleotide in position 2058, which is crucial for macrolides binding, plays a major role in macrolide selectivity.…”

“…However, careful comparisons between the antibiotic-binding sites in ribosomes from the eubacterium D. Radiodurans and those from the archaeon H. marismortui highlighted a neat distinction in the nucleotide sequence and orientation, leading to substantial differences in macrolide binding modes [2,75,80]. Hence, it was concluded that although A2058 is the main macrolide binding 'anchor', it is not the sole nucleotide determining drug positioning and, therefore, effectiveness.…”