Bacteria growing in different conditions experience a broad range of demand on the rate of protein synthesis which profoundly affects cellular resource allocation. During fast growth, protein synthesis is long known to be modulated by adjusting the ribosome content, with the vast majority of ribosomes engaged at a near-maximal rate of elongation. Here we characterized protein synthesis by E. coli systematically, focusing on slow growth conditions. We establish that the translational elongation rate decreases as growth slows down, exhibiting a Michaelis-Menten dependence on the abundance of the cellular translational apparatus. However, an appreciable elongation rate is maintained even towards zero growth including the stationary phase. This maintenance, critical for timely protein synthesis in harsh environments, is accompanied by a drastic reduction in the fraction of active ribosomes. Interestingly, well-known antibiotics such as chloramphenicol also cause substantial reduction in the pool of active ribosomes, instead of slowing down translational elongation as commonly thought.

Whereas DNA viruses are known to be abundant, diverse, and commonly key ecosystem players, RNA viruses are insufficiently studied outside disease settings. In this study, we analyzed ≈28 terabases of Global Ocean RNA sequences to expand Earth’s RNA virus catalogs and their taxonomy, investigate their evolutionary origins, and assess their marine biogeography from pole to pole. Using new approaches to optimize discovery and classification, we identified RNA viruses that necessitate substantive revisions of taxonomy (doubling phyla and adding >50% new classes) and evolutionary understanding. “Species”-rank abundance determination revealed that viruses of the new phyla “ Taraviricota ,” a missing link in early RNA virus evolution, and “ Arctiviricota ” are widespread and dominant in the oceans. These efforts provide foundational knowledge critical to integrating RNA viruses into ecological and epidemiological models.

A widely held view is that directional movement of tRNA in the ribosome is determined by an intrinsic mechanism and driven thermodynamically by transpeptidation. Here, we show that, in certain ribosomal complexes, the pretranslocation (PRE) state is thermodynamically favored over the posttranslocation (POST) state. Spontaneous and efficient conversion from the POST to PRE state is observed when EF-G is depleted from ribosomes in the POST state or when tRNA is added to the E site of ribosomes containing P-site tRNA. In the latter assay, the rate of tRNA movement is increased by streptomycin and neomycin, decreased by tetracycline, and not affected by the acylation state of the tRNA. In one case, we provide evidence that complex conversion occurs by reverse translocation (i.e., direct movement of the tRNAs from the E and P sites to the P and A sites, respectively). These findings have important implications for the energetics of translocation.

The widely used antibiotic spectinomycin inhibits bacterial protein synthesis by blocking translocation of messenger RNA and transfer RNAs on the ribosome. Here, we show that in crystals of the Escherichia coli 70S ribosome spectinomycin binding traps a distinct swiveling state of the head domain of the small ribosomal subunit. Spectinomycin also alters the rate and completeness of reverse translocation in vitro. These structural and biochemical data indicate that in solution spectinomycin sterically blocks swiveling of the head domain of the small ribosomal subunit and thereby disrupts the translocation cycle.Spectinomycin, a broad-spectrum antibiotic that selectively targets bacterial ribosomes (1-3), remains important in clinical and veterinary use, as it induces only low levels of bacterial resistance (4-6). Despite decades of worldwide use of the drug, the molecular mechanism by which spectinomycin inhibits translation remains unclear.Spectinomycin, an aminocyclitol constituted from two glucose moieties (7) (Figure 1, panel a), inhibits translocation of transfer RNAs (tRNAs) and messenger RNA (mRNA) on the ribosome (8-10), the step that follows the formation of each peptide bond. Translocation, catalyzed by the GTPase elongation factor G (EF-G), involves multiple large conformational * Corresponding author, jcate@lbl.gov.. Supporting Information Available:This material is available free of charge via the Internet.Accession Codes: The coordinates and structure factors have been deposited in the Protein Data Bank with accession codes 2QOU, 2QOV, 2QOW, and 2QOX (70S ribosome in complex with spectinomycin), and 2QOY, 2QOZ, 2QP0, and 2QP1 (70S ribosome in complex with spectinomycin and neomycin). HHS Public Access Author ManuscriptAuthor Manuscript Author ManuscriptAuthor Manuscript changes in the ribosome (9,(11)(12)(13)(14)(15). Translocation is thought to begin with a ratchet-like motion of the small (30S) ribosomal subunit relative to the large (50S) ribosomal subunit (11,15,16), followed by swiveling of the head of the small subunit (12, 13) and "unlocking" (opening) of the tRNA binding groove to allow peptidyl (P-site) tRNA to pass into the Exit (E) site (13,14,17,18). These conformational changes lead to discrete movements of bound tRNAs, where the tRNAs move first with respect to the 50S subunit into hybrid binding states and then move with respect to the 30S subunit (19). Unlike most of the translocation inhibitors, spectinomycin slows but does not completely abolish multiple-turnover translocation (9). It also does not induce mRNA miscoding (20, 21).The binding site for spectinomycin, in RNA helix 34 (h34) of the head domain of the 30S subunit, resides near the single ribosomal RNA (rRNA) helix that connects the head domain to the rest of the small subunit (i.e., neck of the 30S subunit) and faces the mRNA binding groove (22-26) (Figure 1, panels b and c; Supplementary Figure 1). Biochemical data (9,24,25,27) and the structure of spectinomycin bound to the 30S ribosomal subunit (26) suggested ...

During protein synthesis, transfer RNAs (tRNAs) are translocated from the aminoacyl to peptidyl to exit sites of the ribosome, coupled to the movement of messenger RNA (mRNA), in a reaction catalyzed by elongation factor G (EF-G) and guanosine triphosphate (GTP). Here, we show that the peptidyl transferase inhibitor sparsomycin triggers accurate translocation in vitro in the absence of EF-G and GTP. Our results provide evidence that translocation is a function inherent to the ribosome and that the energy to drive this process is stored in the tRNA-mRNA-ribosome complex after peptide-bond formation. These findings directly implicate the peptidyl transferase center of the 50S subunit in the mechanism of translocation, a process involving large-scale movement of tRNA and mRNA in the 30S subunit, some 70 angstroms away.

Sixty-one codons specify 20 amino acids offering cells many options for encoding a polypeptide sequence. Two new studies (Cannarozzi et al, 2010, Tuller et al., 2010) now foster the idea that patterns of codon usage can control ribosome speed, fine-tuning translation to increase the efficiency of protein synthesis.

The molecular basis of the induced-fit mechanism that determines the fidelity of protein synthesis remains unclear. Here, we isolated mutations in 16S rRNA that increase the rate of miscoding and stop codon read-through. Many of the mutations clustered along interfaces between the 30S shoulder domain and other parts of the ribosome, strongly implicating shoulder movement in the induced-fit mechanism of decoding. The largest subset of mutations mapped to helices h8 and h14. These helices interact with each other and with the 50S subunit to form bridge B8. Previous cryo-EM studies revealed a contact between h14 and the switch 1 motif of EF-Tu, raising the possibility that h14 plays a direct role in GTPase activation. To investigate this possibility, we constructed both deletions and insertions in h14. While ribosomes harboring a 2-base-pair (bp) insertion in h14 were completely inactive in vivo, those containing a 2-bp deletion retained activity but were error prone. In vitro, the truncation of h14 accelerated GTP hydrolysis for EF-Tu bearing near-cognate aminoacyl-tRNA, an effect that can largely account for the observed miscoding in vivo. These data show that h14 does not help activate EF-Tu but instead negatively controls GTP hydrolysis by the factor. We propose that bridge B8 normally acts to counter inward rotation of the shoulder domain; hence, mutations in h8 and h14 that compromise this bridge decrease the stringency of aminoacyl-tRNA selection.

During translation, tRNAs must move rapidly to their adjacent sites in the ribosome while maintaining precise pairing with mRNA. This movement (translocation) occurs in a stepwise manner with hybrid-state intermediates, but it is unclear how these hybrid states relate to kinetically defined events of translocation. Here we analyze mutations at position 2394 of 23S rRNA in a pre-steadystate kinetic analysis of translocation. These mutations target the 50S E site and are predicted to inhibit P/E state formation. Each mutation decreases growth rate, the maximal rate of translocation (k trans), and the apparent affinity of EF-G for the pretranslocation complex (i.e., increases K 1/2). The magnitude of these defects follows the trend A > G > U. Because the C2394A mutation did not decrease the rate of single-turnover GTP hydrolysis, the >20-fold increase in K 1/2 conferred by C2394A can be attributed to neither the initial binding of EF-G nor the subsequent GTP hydrolysis step. We propose that C2394A inhibits a later step, P/E state formation, to confer its effects on translocation. Replacement of the peptidyl group with an aminoacyl group, which is predicted to inhibit A/P state formation, decreases k trans without increasing K1/2. These data suggest that movements of tRNA into the P/E and A/P sites are separable events. This mutational study allows tRNA movements with respect to both subunits to be integrated into a kinetic model for translocation.elongation factor G ͉ translation ͉ exit site ͉ GTPase M ovement of tRNAs through the ribosome is believed to occur in a stepwise manner (1). Chemical protection experiments showed that, after peptidyl transfer, the acceptor ends of the tRNAs can move spontaneously with respect to the 50S subunit to form the hybrid state. In the hybrid state, the deacylated tRNA occupies the 30S P site and 50S E site (P/E site) while the peptidyl-tRNA occupies the 30S A site and 50S P site (A/P site). It was proposed that hybrid state formation precedes codon-anticodon movement within the 30S subunit, and only the latter event requires catalysis by elongation factor G (EF-G). Support for the hybrid-state model came from Förster resonance energy transfer (FRET) experiments in which an Ϸ20-Å movement of the 5Ј end of the newly deacylated tRNA toward ribosomal protein L1 was inferred after peptide bond formation, whereas the position of the peptidyl group changed little (2). Because L1 lies near the 50S E site, these data are consistent with movement of tRNA into the P/E site. Further evidence came from single-molecule FRET studies that monitored the distance between probes attached to the elbows of tRNAs in the A and P sites. Fluctuations between two distinct configurations were observed that were structurally consistent with the classical and hybrid states (3). More recently, a third state of the pretranslocation (PRE) complex was observed by single-molecule FRET, consistent with one tRNA bound to the P/E site and the other bound to the A/A site (4). Experiments that monitored mRNA in ribos...