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.

The ribosome and protein synthesis are major targets within the cell for inhibition by antibiotics, such as the tetracyclines. The tetracycline family of antibiotics represent a large and diverse group of compounds, ranging from the naturally produced chlortetracycline, introduced into medical usage in the 1940s, to second and third generation semi-synthetic derivatives of tetracycline, such as doxycycline, minocycline and more recently the glycylcycline tigecycline. Here we describe the mode of interaction of tetracyclines with the ribosome and mechanism of action of this class of antibiotics to inhibit translation. Additionally, we provide an overview of the diverse mechanisms by which bacteria obtain resistance to tetracyclines, ranging from efflux, drug modification, target mutation and the employment of specialized ribosome protection proteins.

During translation, transfer RNAs enter the ribosome and then move sequentially through three sites, known as A, P and E, as they transfer their attached amino acids onto the growing peptide chain. How the ribosome facilitates tRNA translocation between the sites remains largely unknown. Christian Spahn and colleagues have used multiparticle cryoelectron microscopy of a ribosome bound to the translation elongation factor, EF-G, to get information about tRNA movement. They identify two new sub-states and conclude that, following spontaneous inter-subunit ratcheting, translocation is the direct result of head swivelling and unratcheting of the 30S ribosomal subunit. During translation, tRNAs enter the ribosome and then move sequentially through three sites, known as A, P and E, as they transfer their attached amino acids onto the growing peptide chain. How the ribosome facilitates tRNA translocation between the sites remains largely unknown. Now a study uses multiparticle cryoelectron microscopy of a ribosome bound to the translation elongation factor, EF-G, to get information about tRNA movement. It identifies two new substates and sees that translocation is linked to unratcheting of the 30S ribosomal subunit. 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 site1,2. The translocation reaction is catalysed by elongation factor G (EF-G) in a GTP-dependent manner3. 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.

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 theDeinococcus radiodurans50S 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.

Significance: During protein synthesis, ribosomes catalyze peptide-bond formation between amino acids with differing efficiency. We show that two or more consecutive prolines induce ribosome stalling, and that stalling strength is influenced by the amino acid preceding and following the prolines. In bacteria, the elongation factor EF-P efficiently rescues the ribosome stalling irrespective of the XPP or PPX motif.