Daniel N. Wilson

Ribosome-targeting antibiotics and mechanisms of bacterial resistance

The ribosome is one of the main antibiotic targets in the bacterial cell. Crystal structures of naturally produced antibiotics and their semi-synthetic derivatives bound to ribosomal particles have provided unparalleled insight into their mechanisms of action, and they are also facilitating the design of more effective antibiotics for targeting multidrug-resistant bacteria. In this Review, I discuss the recent structural insights into the mechanism of action of ribosome-targeting antibiotics and the molecular mechanisms of bacterial resistance, in addition to the approaches that are being pursued for the production of improved drugs that inhibit bacterial protein synthesis.

Structures of the human and Drosophila 80S ribosome

Protein synthesis in all cells is carried out by macromolecular machines called ribosomes. Although the structures of prokaryotic, yeast and protist ribosomes have been determined, the more complex molecular architecture of metazoan 80S ribosomes has so far remained elusive. Here we present structures of Drosophila melanogaster and Homo sapiens 80S ribosomes in complex with the translation factor eEF2, E-site transfer RNA and Stm1-like proteins, based on high-resolution cryo-electron-microscopy density maps. These structures not only illustrate the co-evolution of metazoan-specific ribosomal RNA with ribosomal proteins but also reveal the presence of two additional structural layers in metazoan ribosomes, a well-ordered inner layer covered by a flexible RNA outer layer. The human and Drosophila ribosome structures will provide the basis for more detailed structural, biochemical and genetic experiments.

Head swivel on the ribosome facilitates translocation by means of intra-subunit tRNA hybrid sites

The elongation cycle of protein synthesis involves the delivery of aminoacyl-transfer RNAs to the aminoacyl-tRNA-binding site (A site) of the ribosome, followed by peptide-bond formation and translocation of the tRNAs through the ribosome to reopen the A site. The translocation reaction is catalysed by elongation factor G (EF-G) in a GTP-dependent manner. Despite the availability of structures of various EF-G–ribosome complexes, the precise mechanism by which tRNAs move through the ribosome still remains unclear. Here we use multiparticle cryoelectron microscopy analysis to resolve two previously unseen subpopulations within Thermus thermophilus EF-G–ribosome complexes at subnanometre resolution, one of them with a partly translocated tRNA. Comparison of these substates reveals that translocation of tRNA on the 30S subunit parallels the swivelling of the 30S head and is coupled to unratcheting of the 30S body. Because the tRNA maintains contact with the peptidyl-tRNA-binding site (P site) on the 30S head and simultaneously establishes interaction with the exit site (E site) on the 30S platform, a novel intra-subunit ‘pe/E’ hybrid state is formed. This state is stabilized by domain IV of EF-G, which interacts with the swivelled 30S-head conformation. These findings provide direct structural and mechanistic insight into the ‘missing link’ in terms of tRNA intermediates involved in the universally conserved translocation process.

Structural Insight Into Nascent Polypeptide Chain-Mediated Translational Stalling

Nascent Chains Revealed Detailed analysis of protein translation and translocation across membranes requires the identification and structural analysis of intermediates involved in these processes (see the Perspective by Kampmann and Blobel). Seidelt et al. (p. 1412, published online 29 October) report the visualization by cryo-electron microscopy of a nascent polypeptide chain in the tunnel of the ribosome at 5.8 angstroms. This resolution allows analysis of the conformation and distinct contacts of the nascent chain within the ribosomal tunnel, which suggests a mechanism by which translational stalling is induced by this peptide. Protein translocation across cellular membranes involves the Sec61 protein, a component of a protein-conducting channel. Whether Sec61 acts as a monomer or as an oligomer during protein translocation has been unclear. Becker et al. (p. 1369, published online 29 October) describe active yeast and mammalian ribosome-Sec61 structures that show the Sec61 complex interacting with the ribosome and a nascent secretory protein signal sequence. The analysis unambiguously reveals that the active protein-conducting channel is a single Sec61 copy with its central pore serving as conduit for the nascent polypeptide. Individual polypeptide nascent chains can adopt distinct conformations within the ribosome exit tunnel. Expression of the Escherichia coli tryptophanase operon depends on ribosome stalling during translation of the upstream TnaC leader peptide, a process for which interactions between the TnaC nascent chain and the ribosomal exit tunnel are critical. We determined a 5.8 angstrom–resolution cryo–electron microscopy and single-particle reconstruction of a ribosome stalled during translation of the tnaC leader gene. The nascent chain was extended within the exit tunnel, making contacts with ribosomal components at distinct sites. Upon stalling, two conserved residues within the peptidyltransferase center adopted conformations that preclude binding of release factors. We propose a model whereby interactions within the tunnel are relayed to the peptidyltransferase center to inhibit translation. Moreover, we show that nascent chains adopt distinct conformations within the ribosomal exit tunnel.

Translation elongation factor EF-P alleviates ribosome stalling at polyproline stretches.

Translating Polyproline Translation of messenger RNA into protein is carried out by the ribosome, together with a variety of accessory factors, which offer the potential for regulation of this critical step in gene expression (see the Perspective by Buskirk and Green). Ude et al. (p. 82, published online 13 December), using bacterial genetics and an in vitro reconstituted translation system, and Doerfel et al. (p. 85, published online 13 December), using a model assay for peptide bond formation, find that the universally conserved bacterial elongation factor P (EF-P) (which is orthologous to the archaeal and eukaryotic initiation factor 5A) is required for the efficient translation of polyproline-containing polypeptides. Such short polyproline stretches (with runs of two, three, or more proline residues) would otherwise cause ribosomal stalling. A universally conserved translation factor facilitates synthesis of peptides that would otherwise cause ribosome stalling. [Also see Perspective by Buskirk and Green] Translation elongation factor P (EF-P) is critical for virulence in bacteria. EF-P is present in all bacteria and orthologous to archaeal and eukaryotic initiation factor 5A, yet the biological function has so far remained enigmatic. Here, we demonstrate that EF-P is an elongation factor that enhances translation of polyproline-containing proteins: In the absence of EF-P, ribosomes stall at polyproline stretches, whereas the presence of EF-P alleviates the translational stalling. Moreover, we demonstrate the physiological relevance of EF-P to fine-tune the expression of the polyproline-containing pH receptor CadC to levels necessary for an appropriate stress response. Bacterial, archaeal, and eukaryotic cells have hundreds to thousands of polyproline-containing proteins of diverse function, suggesting that EF-P and a/eIF-5A are critical for copy-number adjustment of multiple pathways across all kingdoms of life.

Dissection of the Mechanism for the Stringent Factor RelA

During conditions of nutrient deprivation, ribosomes are blocked by uncharged tRNA at the A site. The stringent factor RelA binds to blocked ribosomes and catalyzes synthesis of (p)ppGpp, a secondary messenger that induces the stringent response. We demonstrate that binding of RelA and (p)ppGpp synthesis are inversely coupled, i.e., (p)ppGpp synthesis decreases the affinity of RelA for the ribosome. RelA binding to ribosomes is governed primarily by mRNA, but independently of ribosomal protein L11, while (p)ppGpp synthesis strictly requires uncharged tRNA at the A site and the presence of L11. A model is proposed whereby RelA hops between blocked ribosomes, providing an explanation for how low intracellular concentrations of RelA (1/200 ribosomes) can synthesize (p)ppGpp at levels that accurately reflect the starved ribosome population.

The A-Z of bacterial translation inhibitors

Protein synthesis is one of the major targets in the cell for antibiotics. This review endeavors to provide a comprehensive “post-ribosome structure” A–Z of the huge diversity of antibiotics that target the bacterial translation apparatus, with an emphasis on correlating the vast wealth of biochemical data with more recently available ribosome structures, in order to understand function. The binding site, mechanism of action, and modes of resistance for 26 different classes of protein synthesis inhibitors are presented, ranging from ABT-773 to Zyvox. In addition to improving our understanding of the process of translation, insight into the mechanism of action of antibiotics is essential to the development of novel and more effective antimicrobial agents to combat emerging bacterial resistance to many clinically-relevant drugs.

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. Binding of linezolid stabilizes a distinct conformation of the universally conserved 23S rRNA nucleotide U2585 that would be nonproductive for peptide bond formation. In conjunction with available biochemical data, we present a model whereby oxazolidinones impart their inhibitory effect by perturbing the correct positioning of tRNAs on the ribosome.

The highly conserved LepA is a ribosomal elongation factor that back-translocates the ribosome.

The ribosomal elongation cycle describes a series of reactions prolonging the nascent polypeptide chain by one amino acid and driven by two universal elongation factors termed EF-Tu and EF-G in bacteria. Here we demonstrate that the extremely conserved LepA protein, present in all bacteria and mitochondria, is a third elongation factor required for accurate and efficient protein synthesis. LepA has the unique function of back-translocating posttranslocational ribosomes, and the results suggest that it recognizes ribosomes after a defective translocation reaction and induces a back-translocation, thus giving EF-G a second chance to translocate the tRNAs correctly. We suggest renaming LepA as elongation factor 4 (EF4).

Ribosomal Proteins in the Spotlight

ABSTRACT The assignment of specific ribosomal functions to individual ribosomal proteins is difficult due to the enormous cooperativity of the ribosome; however, important roles for distinct ribosomal proteins are becoming evident. Although rRNA has a major role in certain aspects of ribosomal function, such as decoding and peptidyl-transferase activity, ribosomal proteins are nevertheless essential for the assembly and optimal functioning of the ribosome. This is particularly true in the context of interactions at the entrance pore for mRNA, for the translation-factor binding site and at the tunnel exit, where both chaperones and complexes associated with protein transport through membranes bind.