Differences between the structures of bacterial, archaeal, and eukaryotic ribosomes account for the selective action of antibiotics. Even minor variations in the structure of ribosomes of different bacterial species may lead to idiosyncratic, species-specific interactions of the drugs with their targets. Although crystallographic structures of antibiotics bound to the peptidyl transferase center or the exit tunnel of archaeal ( Haloarcula marismortui ) and bacterial ( Deinococcus radiodurans ) large ribosomal subunits have been reported, it remains unclear whether the interactions of antibiotics with these ribosomes accurately reflect those with the ribosomes of pathogenic bacteria. Here we report X-ray crystal structures of the Escherichia coli ribosome in complexes with clinically important antibiotics of four major classes, including the macrolide erythromycin, the ketolide telithromycin, the lincosamide clindamycin, and a phenicol, chloramphenicol, at resolutions of ∼3.3  Å –3.4  Å . Binding modes of three of these antibiotics show important variations compared to the previously determined structures. Biochemical and structural evidence also indicates that interactions of telithromycin with the E. coli ribosome more closely resembles drug binding to ribosomes of bacterial pathogens. The present data further argue that the identity of nucleotides 752, 2609, and 2055 of 23S ribosomal RNA explain in part the spectrum and selectivity of antibiotic action.

During protein synthesis, the ribosome controls the movement of transfer RNA (tRNA) and messenger RNA (mRNA) by means of large-scale structural rearrangements. We describe structures of the intact bacterial ribosome from Escherichia coli that reveal how the ribosome binds tRNA in two functionally distinct states, determined to a resolution of ~3.2 Å by x-ray crystallography. One state positions tRNA in the peptidyl-tRNA binding site. The second, a fully rotated state, is stabilized by ribosome recycling factor (RRF) and binds tRNA in a highly bent conformation in a hybrid peptidyl/exit (P/E) site. The structures help to explain how the ratchet-like motion of the two ribosomal subunits contributes to the mechanisms of translocation, termination, and ribosome recycling.

SummaryStructures of the E. coli 70S ribosome show how the large and small subunits rotate to facilitate protein synthesis.Protein biosynthesis on the ribosome requires repeated cycles of ratcheting, which couples rotation of the two ribosomal subunits with respect to each other and swiveling of the head domain of the small subunit. However, the molecular basis for how the two ribosomal subunits rearrange contacts with each other during ratcheting while remaining stably associated is not known. Here we describe x-ray crystal structures of the intact Escherichia coli ribosome, either in the apo form (3.5 Å resolution) or with one (4.0 Å res) or two (4.0 Å res) anticodon stem-loop tRNA mimics bound, that reveal intermediate states of intersubunit rotation. In the structures, the interface between the small and large ribosomal subunits rearranges in discrete steps along the ratcheting pathway. Positioning of the head domain of the small subunit is controlled by interactions with the large subunit and with the tRNA bound in the peptidyl-tRNA site. The intermediates observed here provide insight into how tRNAs move into the hybrid state of binding that precedes the final steps of mRNA and tRNA translocation.Protein biosynthesis requires many large-scale rearrangements in the ribosome as each amino acid is added to a growing polypeptide chain. Positioning of tRNA on the ribosome is proposed to occur through a ratcheting mechanism. Central to this mechanism is a rotation of the small ribosomal subunit relative to the large subunit (Fig. 1A) (1,2) that occurs in all stages of translation-initiation, elongation, termination, and ribosome recycling (1)-and is targeted by clinically useful antibiotics (3,4). For example after each peptide bond is formed, an ~8°i ntersubunit rotation results in tRNAs bound in the aminoacyl-tRNA and peptidyl-tRNA binding sites (A site and P site, respectively) moving into the P site and exit-tRNA site (E site) on the large ribosomal subunit (Fig. 1B). From this hybrid state of tRNA binding (Fig. 1B) (1,5), the tRNAs are then translocated to the P site and E site on the small subunit.In addition to intersubunit rotation, ratcheting also involves a nearly orthogonal rotation of the head domain of the small ribosomal subunit (Fig. 1C) that plays a role in controlling the position of tRNAs within the ribosome (1,6,7). As with intersubunit rotation, movement of the head domain is a target for clinically useful antibiotics (8). Swiveling of the head domain relative to the body of the small subunit may also be required for the intrinsic helicase activity of the ribosome in unwinding secondary structure in mRNA (8,9). Rotations of up to 14° allow the head domain to change its position by 20 Å or more at the ribosomal subunit interface, or the width of a tRNA substrate (7). The molecular basis for how the ribosomal subunits rotate with respect to each other while remaining stably associated remains unknown (1,10). Furthermore, the precise timing of movements of the small subunit head domain during ra...

The biological functions of RNA are ultimately governed by the local environment at each nucleotide. Selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) chemistry is a powerful approach for measuring nucleotide structure and dynamics in diverse biological environments. SHAPE reagents acylate the 2′-hydroxyl group at flexible nucleotides because unconstrained nucleotides preferentially sample rare conformations that enhance the nucleophilicity of the 2′-hydroxyl. The critical corollary is that some constrained nucleotides must be poised for efficient reaction at the 2′-hydroxyl group. To identify such nucleotides, we performed SHAPE on intact crystals of the E. coli ribosome, monitored the reactivity of 1490 nucleotides in 16S ribosomal RNA, and examined those nucleotides that were hyper-reactive towards SHAPE and had well-defined crystallographic conformations. Analysis of these conformations revealed that 2′-hydroxyl reactivity is broadly facilitated by general base catalysis involving multiple RNA functional groups and by two specific orientations of the bridging 3′-phosphate group. Nucleotide analog studies confirmed the contributions of these mechanisms to SHAPE reactivity. These results provide a strong mechanistic explanation for the relationship between SHAPE reactivity and local RNA dynamics and will facilitate interpretation of SHAPE information in the many technologies that make use of this chemistry.

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