Search

Many antibiotics that bind to the ribosome inhibit translation by blocking the movement of tRNAs and mRNA or interfering with ribosome dynamics, which impairs the formation of essential translocation intermediates. Here we show how translocation inhibitors viomycin (Vio), neomycin (Neo), paromomycin (Par), kanamycin (Kan), spectinomycin (Spc), hygromycin B (HygB), and streptomycin (Str, an antibiotic that does not inhibit tRNA movement), affect principal motions of the small ribosomal subunits (SSU) during EF-G-promoted translocation. Using ensemble kinetics, we studied the SSU body domain rotation and SSU head domain swiveling in real time. We show that although antibiotics binding to the ribosome can favor a particular ribosome conformation in the absence of EF-G, their kinetic effect on the EF-G-induced transition to the rotated/swiveled state of the SSU is moderate. The antibiotics mostly inhibit backward movements of the SSU body and/or the head domains. Vio, Spc, and high concentrations of Neo completely inhibit the backward movements of the SSU body and head domain. Kan, Par, HygB, and low concentrations of Neo slow down both movements, but their sequence and coordination are retained. Finally, Str has very little effect on the backward rotation of the SSU body domain, but retards the SSU head movement. The data underscore the importance of ribosome dynamics for tRNA-mRNA translocation and provide new insights into the mechanism of antibiotic action.

Viomycin, an antibiotic that has been used to fight tuberculosis infections, is believed to block the translocation step of protein synthesis by inhibiting ribosomal subunit dissociation and trapping the ribosome in an intermediate state of intersubunit rotation. The mechanism by which viomycin stabilizes this state remains unexplained. To address this, we have determined cryo-EM and X-ray crystal structures of Escherichia coli 70S ribosome complexes trapped in a rotated state by viomycin. The 3.8-Å resolution cryo-EM structure reveals a ribosome trapped in the hybrid state with 8.6° intersubunit rotation and 5.3° rotation of the 30S subunit head domain, bearing a single P/E state transfer RNA (tRNA). We identify five different binding sites for viomycin, four of which have not been previously described. To resolve the details of their binding interactions, we solved the 3.1-Å crystal structure of a viomycin-bound ribosome complex, revealing that all five viomycins bind to ribosomal RNA. One of these (Vio1) corresponds to the single viomycin that was previously identified in a complex with a nonrotated classical-state ribosome. Three of the newly observed binding sites (Vio3, Vio4, and Vio5) are clustered at intersubunit bridges, consistent with the ability of viomycin to inhibit subunit dissociation. We propose that one or more of these same three viomycins induce intersubunit rotation by selectively binding the rotated state of the ribosome at dynamic elements of 16S and 23S rRNA, thus, blocking conformational changes associated with molecular movements that are required for translocation.

The growth of the polypeptide chain occurs due to the fast and coordinated work of the ribosome and protein elongation factors, EF-Tu and EF-G. However, the exact contribution of each of these components in the overall balance of translation kinetics remains not fully understood. We created an in vitro translation system Escherichia coli replacing either elongation factor with heterologous thermophilic protein from Thermus thermophilus. The rates of the A-site binding and decoding reactions decreased an order of magnitude in the presence of thermophilic EF-Tu, indicating that the kinetics of aminoacyl-tRNA delivery depends on the properties of the elongation factor. On the contrary, thermophilic EF-G demonstrated the same translocation kinetics as a mesophilic protein. Effects of translocation inhibitors (spectinomycin, hygromycin B, viomycin and streptomycin) were also similar for both proteins. Thus, the process of translocation largely relies on the interaction of tRNAs and the ribosome and can be efficiently catalysed by thermophilic EF-G even at suboptimal temperatures.

Many antibiotics that bind to the ribosome inhibit translation by blocking the movement of tRNAs and mRNA or interfering with ribosome dynamics, which impairs the formation of essential translocation intermediates. Here we show how translocation inhibitors viomycin (Vio), neomycin (Neo), paromomycin (Par), kanamycin (Kan), spectinomycin (Spc), hygromycin B (HygB), and streptomycin (Str, an antibiotic that does not inhibit tRNA translocation), affect principal motions of the small ribosomal subunits (SSU) during EF-G-promoted translocation. Using ensemble kinetics, we studied the SSU body domain rotation and SSU head domain swiveling in real time. We show that although antibiotics binding to the ribosome can favor a particular ribosome conformation in the absence of EF-G, EF-G-induced transition to the rotated/swiveled state of the SSU is hardly affected. The major effect of the antibiotics is observed at the stage when the SSU body and the head domain move backward. Vio, Spc and high concentrations of Neo completely inhibit the backward movements of the SSU body and head domain. Kan, Par, HygB and low concentrations of Neo slow down both movements, but their sequence and coordination are retained. Finally, Str has very little effect on the backward rotation of the SSU body domain, but retards the SSU head movement. The data underscore the importance of ribosome dynamics for tRNA-mRNA translocation and provide new insights into the mechanism of antibiotic action.

Bacterial ribosome recycling requires breakdown of the post-termination complex (PoTC), comprising a messenger RNA (mRNA) and an uncharged transfer RNA (tRNA) cognate to the terminal mRNA codon bound to the 70S ribosome. The translation factors, elongation factor G and ribosome recycling factor, are known to be required for recycling, but there is controversy concerning whether these factors act primarily to effect the release of mRNA and tRNA from the ribosome, with the splitting of the ribosome into subunits being somewhat dispensable, or whether their main function is to catalyze the splitting reaction, which necessarily precedes mRNA and tRNA release. Here, we utilize three assays directly measuring the rates of mRNA and tRNA release and of ribosome splitting in several model PoTCs. Our results largely reconcile these previously held views. We demonstrate that, in the absence of an upstream Shine–Dalgarno (SD) sequence, PoTC breakdown proceeds in the order: mRNA release followed by tRNA release and then by 70S splitting. By contrast, in the presence of an SD sequence all three processes proceed with identical apparent rates, with the splitting step likely being rate-determining. Our results are consistent with ribosome profiling results demonstrating the influence of upstream SD-like sequences on ribosome occupancy at or just before the mRNA stop codon.

The ribosome is one of the major targets in the cell for clinically used antibiotics. However, the increase in multidrug resistant bacteria is rapidly reducing the effectiveness of our current arsenal of ribosome-targeting antibiotics, highlighting the need for the discovery of compounds with new scaffolds that bind to novel sites on the ribosome. One possible avenue for the development of new antimicrobial agents is by characterization and optimization of ribosome-targeting peptide antibiotics. Biochemical and structural data on ribosome-targeting peptide antibiotics illustrates the large diversity of scaffolds, binding interactions with the ribosome as well as mechanism of action to inhibit translation. The availability of high-resolution structures of ribosomes in complex with peptide antibiotics opens the way to structure-based design of these compounds as novel antimicrobial agents.

The unique advantage of the single molecule approach is to reveal the inhomogeneous subpopulations in an ensemble. For example, smFRET (single molecule fluorescence resonance energy transfer) can identify multiple subpopulations based on the FRET efficiency histograms. However, identifying multiple FRET states with overlapping average values remains challenging. Here, we report a new concept and method to analyze the single molecule FRET data of a ribosome system. The main results are as follows: 1. based on a hierarchic concept, multiple ribosome subpopulations are identified. 2. The subpopulations are self-identified via the cross-correlation analysis of the FRET histogram profiles. The dynamic heterogeneity is tracked after 2 min intervals on the same ribosomes individually. 3. The major ribosome subpopulations exchange with each other with a certain pattern, indicating some correlations among the motions of the tRNAs and the ribosomal components. Experiments under the conditions of 20% glycerol or 1mM viomycin supported this conclusion.

The cytotoxin colicin E3 targets the 30S subunit of bacterial ribosomes and specifically cleaves 16S rRNA at the decoding centre, thereby inhibiting translation. Although the cleavage site is well known, it is not clear which step of translation is inhibited. We studied the effects of colicin E3 cleavage on ribosome functions by analysing individual steps of protein synthesis. We find that the cleavage affects predominantly the elongation step. The inhibitory effect of colicin E3 cleavage originates from the accumulation of sequential impaired decoding events, each of which results in low occupancy of the A site and, consequently, decreasing yield of elongating peptide. The accumulation leads to an almost complete halt of translation after reading of a few codons. The cleavage of 16S rRNA does not impair monitoring of codon–anticodon complexes or GTPase activation during elongation-factor Tu-dependent binding of aminoacyl-tRNA, but decreases the stability of the codon–recognition complex and slows down aminoacyl-tRNA accommodation in the A site. The tRNA–mRNA translocation is faster on colicin E3-cleaved than on intact ribosomes and is less sensitive to inhibition by the antibiotic viomycin.

Viomycin is a tuberactinomycin antibiotic essential for treating multidrug-resistant tuberculosis. It inhibits bacterial protein synthesis by blocking elongation factor G (EF-G) catalyzed translocation of messenger RNA on the ribosome. Here we have clarified the molecular aspects of viomycin inhibition of the elongating ribosome using pre-steady-state kinetics. We found that the probability of ribosome inhibition by viomycin depends on competition between viomycin and EF-G for binding to the pretranslocation ribosome, and that stable viomycin binding requires an A-site bound tRNA. Once bound, viomycin stalls the ribosome in a pretranslocation state for a minimum of ∼ 45 s. This stalling time increases linearly with viomycin concentration. Viomycin inhibition also promotes futile cycles of GTP hydrolysis by EF-G. Finally, we have constructed a kinetic model for viomycin inhibition of EF-G catalyzed translocation, allowing for testable predictions of tuberactinomycin action in vivo and facilitating in-depth understanding of resistance development against this important class of antibiotics.

Capreomycin and the structurally similar compound viomycin are cyclic peptide antibiotics which are particularly active against Mycobacterium tuberculosis , including multidrug resistant strains. Both antibiotics bind across the ribosomal interface involving 23S rRNA helix 69 (H69) and 16S rRNA helix 44 (h44). The binding site of tuberactinomycins in h44 partially overlaps with that of aminoglycosides, and they share with these drugs the side effect of irreversible hearing loss. Here we studied the drug target interaction on ribosomes modified by site-directed mutagenesis. We identified rRNA residues in h44 as the main determinants of phylogenetic selectivity, predict compensatory evolution to impact future resistance development, and propose mechanisms involved in tuberactinomycin ototoxicity, which may enable the development of improved, less-toxic derivatives.

The tuberactinomycin antibiotic family is one of the most effective against multi-drug resistant M. tuberculosis. The structures of two members of this family, viomycin and capreomycin, bound to the ribosome now indicate that they act by stabilizing the A site tRNA in a pre-translocation state and may suggest further avenues for drug development. Viomycin and capreomycin belong to the tuberactinomycin family of antibiotics, which are among the most effective antibiotics against multidrug-resistant tuberculosis. Here we present two crystal structures of the 70S ribosome in complex with three tRNAs and bound to either viomycin or capreomycin at 3.3- and 3.5-A resolution, respectively. Both antibiotics bind to the same site on the ribosome, which lies at the interface between helix 44 of the small ribosomal subunit and helix 69 of the large ribosomal subunit. The structures of these complexes suggest that the tuberactinomycins inhibit translocation by stabilizing the tRNA in the A site in the pretranslocation state. In addition, these structures show that the tuberactinomycins bind adjacent to the binding sites for the paromomycin and hygromycin B antibiotics, which may enable the development of new derivatives of tuberactinomycins that are effective against drug-resistant strains.

Adjacent transfer RNAs (tRNAs) in the A- and P-sites of the ribosome are in dynamic equilibrium between two different conformations called classical and hybrid states before translocation. Here, we have used single-molecule fluorescence resonance energy transfer to study the effect of Mg(2+) on tRNA dynamics with and without an acetyl group on the A-site tRNA. When the A-site tRNA is not acetylated, tRNA dynamics do not depend on [Mg(2+)], indicating that the relative positions of the substrates for peptide-bond formation are not affected by Mg(2+). In sharp contrast, when the A-site tRNA is acetylated, Mg(2+) lengthens the lifetime of the classical state but does not change the lifetime of the hybrid state. Based on these findings, the classical state resembles a state with direct stabilization of tertiary structure by Mg(2+) ions whereas the hybrid state resembles a state with little Mg(2+)-assisted stabilization. The antibiotic viomycin, a translocation inhibitor, suppresses tRNA dynamics, suggesting that the enhanced fluctuations of tRNAs after peptide-bond formation drive spontaneous attempts at translocation by the ribosome.

During protein synthesis, transfer RNA and messenger RNA undergo coupled translocation through the ribosome's A, P and E sites, a process catalyzed by elongation factor EF-G. Viomycin blocks translocation on bacterial ribosomes and is believed to bind at the subunit interface. Using fluorescent resonance energy transfer and chemical footprinting, we show that viomycin traps the ribosome in an intermediate state of translocation. Changes in FRET efficiency show that viomycin causes relative movement of the two ribosomal subunits indistinguishable from that induced by binding of EF-G with GDPNP. Chemical probing experiments indicate that viomycin induces formation of a hybrid-state translocation intermediate. Thus, viomycin inhibits translation through a unique mechanism, locking ribosomes in the hybrid state; the EF-G-induced 'ratcheted' state observed by cryo-EM is identical to the hybrid state; and, since translation is viomycin sensitive, the hybrid state may be present in vivo.

Here we report that the specificity of peptide release in the ribosome on a nonstop mRNA by ArfA and RF2 is achieved by an induced-fit mechanism. Using RF2 that is methylated on the glutamine of its GGQ motif (RF2m), we show that methylation substantially increases the rate of ArfA/RF2-catalyzed peptide release on a nonstop mRNA that does not occupy the ribosomal A site, but has only a modest effect on kcat by the same proteins on longer nonstop mRNAs occupying the A site of the mRNA channel in the ribosome. Our data suggest that enhancement in the kcat of peptide release by ArfA and RF2 under the cognate decoding condition is the result of favorable conformational changes in the nonstop complex. We demonstrate a shared mechanism between canonical and nonstop termination, supported by similarities in the kinetic mechanisms in antibiotic inhibition and methylation-correlated enhancement in the rate of peptide release. Despite these similarities, our data suggest that nonstop termination differs from canonical pathway in the downstream event of recycling.

The cyclic peptide antibiotics capreomycin and viomycin are generally effective against the bacterial pathogen Mycobacterium tuberculosis. However, recent virulent isolates have become resistant by inactivation of their tlyA gene. We show here that tlyA encodes a 2'-O-methyltransferase that modifies nucleotide C1409 in helix 44 of 16S rRNA and nucleotide C1920 in helix 69 of 23S rRNA. Loss of these previously unidentified rRNA methylations confers resistance to capreomycin and viomycin. Many bacterial genera including enterobacteria lack a tlyA gene and the ensuing methylations and are less susceptible than mycobacteria to capreomycin and viomycin. We show that expression of recombinant tlyA in Escherichia coli markedly increases susceptibility to these drugs. When the ribosomal subunits associate during translation, the two tlyA-encoded methylations are brought into close proximity at interbridge B2a. The location of these methylations indicates the binding site and inhibitory mechanism of capreomycin and viomycin at the ribosome subunit interface.

Translocation of mRNA and tRNAs through the ribosome is catalyzed by a universally conserved elongation factor (EF-G in prokaryotes and EF-2 in eukaryotes). Previous studies have suggested that ribosome-bound EF-G undergoes significant structural rearrangements. Here, we follow the movement of domain IV of EF-G, which is critical for the catalysis of translocation, relative to protein S12 of the small ribosomal subunit using single-molecule FRET. We show that ribosome-bound EF-G adopts distinct conformations corresponding to the pre- and posttranslocation states of the ribosome. Our results suggest that, upon ribosomal translocation, domain IV of EF-G moves toward the A site of the small ribosomal subunit and facilitates the movement of peptidyl-tRNA from the A to the P site. We found no evidence of direct coupling between the observed movement of domain IV of EF-G and GTP hydrolysis. In addition, our results suggest that the pretranslocation conformation of the EF-G–ribosome complex is significantly less stable than the posttranslocation conformation. Hence, the structural rearrangement of EF-G makes a considerable energetic contribution to promoting tRNA translocation.

Stringent factor is a ribosome-dependent ATP:GTP pyrophosphoryl transferase that synthesizes (p)ppGpp upon nutrient deprivation. It is activated by unacylated tRNA in the ribosomal amino-acyl site (A-site) but it is unclear how activation occurs. A His-tagged stringent factor was isolated by affinity-chromatography and precipitation. This procedure yielded a protein of high purity that displayed (a) a low endogenous pyrophosphoryl transferase activity that was inhibited by the antibiotic tetracycline; (b) a low ribosome-dependent activity that was inhibited by the A-site specific antibiotics thiostrepton, micrococcin, tetracycline and viomycin; (c) a tRNA- and ribosome-dependent activity amounting to 4500 pmol pppGpp per pmol stringent factor per minute. Footprinting analysis showed that stringent factor interacted with ribosomes that contained tRNAs bound in classical states. Maximal activity was seen when the ribosomal A-site was presaturated with unacylated tRNA. Less tRNA was required to reach maximal activity when stringent factor and unacylated tRNA were added simultaneously to ribosomes, suggesting that stringent factor formed a complex with tRNA in solution that had higher affinity for the ribosomal A-site. However, tRNA-saturation curves, performed at two different ribosome/stringent factor ratios and filter-binding assays, did not support this hypothesis.

Elongation factor G (EF-G) and ribosome recycling factor (RRF) disassemble post-termination complexes of ribosome, mRNA, and tRNA. RRF forms stable complexes with 70 S ribosomes and 50 S ribosomal subunits. Here, we show that EF-G releases RRF from 70 S ribosomal and model post-termination complexes but not from 50 S ribosomal subunit complexes. The release of bound RRF by EF-G is stimulated by GTP analogues. The EF-G-dependent release occurs in the presence of fusidic acid and viomycin. However, thiostrepton inhibits the release. RRF was shown to bind to EF-G-ribosome complexes in the presence of GTP with much weaker affinity, suggesting that EF-G may move RRF to this position during the release of RRF. On the other hand, RRF did not bind to EF-G-ribosome complexes with fusidic acid, suggesting that EF-G stabilized by fusidic acid does not represent the natural post-termination complex. In contrast, the complexes of ribosome, EF-G and thiostrepton could bind RRF, although with lower affinity. These results suggest that thiostrepton traps an intermediate complex having RRF on a position that clashes with the P/E site bound tRNA. Mutants of EF-G that are impaired for translocation fail to disassemble post-termination complexes and exhibit lower activity in releasing RRF. We propose that the release of ribosome-bound RRF by EF-G is required for post-termination complex disassembly. Before release from the ribosome, the position of RRF on the ribosome will change from the original A/P site to a new location that clashes with tRNA on the P/E site.

Ribosomes are dynamic complexes responsible for translating the genetic information encoded in mRNAs to proteins. The accuracy of this process is vital to the survival of an organism, and is often compromised by translational miscoding agents. Aminoglycosides are a group of miscoding agents that bind to the ribosome and reduce the fidelity of translation. Previous studies have shown that aminoglycosides alter the higher order structure of the ribosome. Here, we used a toeprinting assay to how that streptomycin, neomycin, kanamycin, gentamycin, and hygromycin B trigger conformational changes within Escherichia coli ribosome. Miscoding agents viomycin and 30 % ethanol also cause similar structural changes within the ribosome. In contrast, antibiotics that do not cause miscoding, such as tetracycline, chloramphenicol, erythromycin, fusidic acid and spectinomycin, do not induce the conformational changes triggered by miscoding agents. Furthermore, ribosomes isolated from strains that are either streptomycin resistant or dependent for growth do not show these conformational changes in the presence of streptomycin. These results correlate structural changes in the ribosome induced by miscoding agents in vitro with their in vivo phenotype.

One of the most important families of antibiotics are the aminoglycosides, including drugs such as neomycin B, paromomycin, gentamicin and streptomycin. With the discovery of the catalytic potential of RNA, these antibiotics became very popular due to their RNA-binding capacity. They serve for the analysis of RNA function as well as for the study of RNA as a potential therapeutic target. Improvements in RNA structure determination recently provided first insights into the decoding site of the ribosome at high resolution and how aminoglycosides might induce misreading of the genetic code. In addition to inhibiting prokaryotic translation, aminoglycosides inhibit several catalytic RNAs such as self-splicing group I introns, RNase P and small ribozymes in vitro. Furthermore, these antibiotics interfere with human immunodeficiency virus (HIV) replication by disrupting essential RNA-protein contacts. Most exciting is the potential of many RNA-binding antibiotics to stimulate RNA activities, conceiving small-molecule partners for the hypothesis of an ancient RNA world. SELEX (systematic evolution of ligands by exponential enrichment) has been used in this evolutionary game leading to small synthetic RNAs, whose NMR structures gave valuable information on how aminoglycosides interact with RNA, which could possibly be used in applied science.

The class II release factor RF3 is a GTPase related to elongation factor EF-G, which catalyzes release of class I release factors RF1 and RF2 from the ribosome after termination of protein synthesis. The 3.3 Å crystal structure of the RF3·GDPNP·ribosome complex provides a high-resolution description of interactions and structural rearrangements that occur when binding of this translational GTPase induces large-scale rotational movements in the ribosome. RF3 induces a 7° rotation of the body and 14° rotation of the head of the 30S ribosomal subunit, and itself undergoes inter- and intradomain conformational rearrangements. We suggest that ordering of critical elements of switch loop I and the P loop, which help to form the GTPase catalytic site, are caused by interactions between the G domain of RF3 and the sarcin-ricin loop of 23S rRNA. The rotational movements in the ribosome induced by RF3, and its distinctly different binding orientation to the sarcin-ricin loop of 23S rRNA, raise interesting implications for the mechanism of action of EF-G in translocation.

Viomycin belongs to the tuberactinomycin family of antibiotics against tuberculosis. However, its inhibition mechanism remains elusive. Although it is clear that viomycin inhibits the ribosome intersubunit ratcheting, there are contradictory reports about whether the antibiotic viomycin stabilizes the tRNA hybrid or classical state. By using a single-molecule FRET method to directly observe the tRNA dynamics relative to ribosomal protein L27, we have found that viomycin trapped the hybrid state within certain ribosome subgroups but did not significantly suppress the tRNA dynamics. The persistent fluctuation of tRNA implied that tRNA motions were decoupled from the ribosome intersubunit ratcheting. Viomycin also promoted peptidyl-tRNA fluctuation in the posttranslocation complex, implying that, in addition to acylated P-site tRNA, the decoding center also played an important role of ribosome locking after translocation. Therefore, viomycin inhibits translocation by trapping the hybrid state in the pretranslocation complex and disturbing the stability of posttranslocation complex. Our results imply that ribosome translocation is possibly a synergistic process of multiple decoupled local dynamics.

AcPhe2-tRNA(Phe) which appears in ribosomes after consecutive binding of AcPhe-tRNA(Phe) at the P sites and EF-Tu-directed binding of Phe-tRNA(Phe) at the A sites is able to react quantitatively with puromycin in the absence of EF-G. One could readily explain this fact to be the consequence of spontaneous translocation. However, a detailed study of kinetics of puromycin reaction carried out with the use of viomycin (inhibitor of translocation) and the P-site test revealed that, apart from spontaneous translocation, this peptidyl-tRNA could react with puromycin being located at the A site. This leads to the conclusion that the transpeptidation reaction triggers conformational changes in the A-site ribosomal complex bringing the 3'-end of a newly synthesized peptidyl-tRNA nearer to the peptidyl site of peptidyltransferase center. This is detected functionally as a highly pronounced ability of such a peptidyl-tRNA to react with puromycin.

EF4, although structurally similar to the translocase EF-G, promotes back-translocation of tRNAs on the ribosome and is important for bacterial growth under certain conditions. Here, using a coordinated set of in vitro kinetic measures, including changes in the puromycin reactivity of peptidyl-tRNA and in the fluorescence of labeled tRNAs and mRNA, we elucidate the kinetic mechanism of EF4-catalyzed back-translocation and determine the effects of the translocation inhibitors spectinomycin and viomycin on the process. EF4-dependent back-translocation proceeds from a post-translocation (POST) complex to a pre-translocation (PRE) complex via a four-step kinetic scheme (i.e., POST → I1 → I2 → I3 → PRE, which is not the simple reverse of translocation). During back-translocation, movements of the tRNA core regions and of mRNA are closely coupled to one another but are sometimes decoupled from movement of the 3′-end of peptidyl-tRNA. EF4 may be thought of as performing an interrupted catalysis of back-translocation, stopping at the formation of I3 rather than catalyzing the complete process of back-translocation culminating in PRE complex formation. The delay in polypeptide elongation resulting from transient accumulation of I3 is likely to be important for optimizing functional protein biosynthesis.

Aminoglycosides Apramycin, gentamycin, hygromycin B, kanamycin, neomycin, paromomycin, tobramycinElongation (translocation) Most aminoglycoside antibiotics induce translational misreading by promoting binding of near-cognate tRNAs, but the biological effect is probably due to inhibition of the translocation reaction and promotion of back-translocation.Edeine Edeine A Initiation (fMet-tRNA binding) Edeine prevents binding of the initiator tRNA to the 30S subunit in an mRNA-dependent manner.GE81112 GE81112 Initiation (fMet-tRNA binding)GE81112 inhibits binding of the initiator tRNA to the 30S subunit in an mRNA-independent manner.Kasugamycin Kasugamycin Initiation (fMet-tRNA binding) Kasugamycin inhibits translation initiation of canonical mRNAs by binding in the path of the mRNA and preventing stable interaction of the initiator tRNA with the start codon. Pactamycin Pactamycin Initiation, elongation (translocation) Early data suggest that pactamycin allows 30S but not 70S initiation complex formation, whereas recently pactamycin was shown to inhibit translocation in a tRNA-mRNA-dependent manner.Spectinomycin Spectinomycin Elongation (translocation) Spectinomycin binds to the neck of the small subunit and prevents the relative movement of the head and body that is needed for the completion of translocation. Streptomycin Streptomycin Elongation (misreading) Streptomycin increases affinity of tRNA for the A site, has a modest inhibitory effect on translocation, promotes back-translocation, and induces high-level translational misreading.Tetracyclines/glycylcyclines Doxycycline, minocycline, tetracycline/tigecyclineElongation (tRNA delivery) Tetracyclines prevent stable binding of the EF-Tu-tRNA-GTP ternary complex to the ribosome and inhibit accommodation of A-tRNAs upon EF-Tu-dependent GTP hydrolysis.Viomycin Capreomycin, viomycin Elongation (translocation) Viomycin locks tRNAs in a hybrid-site translocation intermediate state, preventing conversion by EF-G into a posttranslocation state.

During protein synthesis transfer RNA (tRNA) and messenger RNA (mRNA) translocate through the ribosome in a rapid and stepwise fashion, a process that is catalyzed by the GTPase elongation factor G (EF-G). Single-molecule fluorescence resonance energy transfer (smFRET) measurements show that the ribosome complex can spontaneously adopt a key translocation intermediate in the absence of nucleotide hydrolysis. By obtaining smFRET data from multiple structural perspectives, we observe that tRNA and EF-G restructure the ribosome energy landscape, increasing the rate at which this intermediate is achieved. In particular, EF-G binding induces a fast dynamic mode of the L1 stalk, which facilitates rapid and reversible formation of the translocation intermediate. Bulk measurements indicate that the extent of conformational coupling is important in determining the rate of translocation. When the extent of coupling is increased, a faster rate of translocation is achieved. The rate of translocation is decreased when the conformational changes are further decoupled, such as by the addition of the antibiotic viomycin. Thus, the extent of coupling of conformational processes on the ribosome may present a new mechanism for the regulation of gene expression.

We describe the optimization of a poly(Phe) synthesis system, the conditions of which have been applied for efficient translation of heteropolymeric mRNAs. Here we identify two parameters that are essential to obtain translation at efficiency and accuracy levels equivalent to those in vivo, viz., the fine-tuning of the energy-rich components with an acetyl-phosphate substrate for energy regeneration, as well as the ionic conditions. Applying this system revealed a number of new features: (i) 70S ribosomes are able to recycle within 300 s in a non-enzymatic fashion in the absence of tmRNA. This observation might explain the fact that a knockout of the tmRNA gene ssrA is not lethal for Escherichia coli cells in contrast to other bacterial strains, such as Bacillus subtilis. (ii) The high efficiency of the system was exploited to analyze the misincorporation of various amino acids (resolution limit=1:15,000). No misreading was observed at the middle codon position and only marginal effects were observed at the first one (even when misreading was artificially stimulated 20- to 30-fold), yielding an improved definition of the near-cognate and non-cognate aminoacyl-tRNAs. (iii) Aminoglycosides increase Phe and Lys incorporation about 2-fold in the presence of poly(U) or poly(UUC) and poly(A), respectively, and induce a back-translocation (except hygromycin B) exclusively in the absence of EF-G*GTP, as do the non-related drugs viomycin and edeine.

Prokaryotic elongation factor G (EF-G) catalyzes the coupled translocations on the ribosome of mRNA and A- and P-site bound tRNAs during the polypeptide elongation cycle. However, the kinetic pathway of this important step has not been fully elucidated. Here we describe the application of single molecule fluorescence resonance energy transfer (smFRET) to probe the mechanism of this key process in real time. Attaching a fluorescent probe within domain IV of EF-G permits monitoring of FRET efficiencies to sites in both ribosomal protein L11 and A-site tRNA. Two different sites on L11, position 38 or position 87, as well as two different A-site tRNAs, Phe-tRNAPhe or Val-tRNAVal were labeled with Cy5. The EF-G:L11 FRET efficiency is constant during EF-G occupancy of the ribosome (300 - 400 ms), whereas the EF-G:tRNA FRET proceeds from high to intermediate to low FRET values. This indicates that EF-G:A-site tRNA distance increases progressively during translocation, with our results providing the first good estimates of the timing of such changes. For both tRNAs, the EF-G:tRNA pair spends most of its time in the intermediate FRET state, with shorter lifetimes in the high and low FRET states. Each transition in the FRET value corresponds to a relative distance change of about 5 to 7 A, so in total, we capture a 12 A relative movement of A-site tRNA away from EF-G during translocation. EF-G:A-site tRNA FRET is sensitive to buffer conditions, with high Mg2+ and polyamines stabilizing the high FRET state. In addition, the translocation inhibitors viomycin and spectinomycin block formation of the low FRET state, although spectinomycin allows more movement between EF-G and tRNA than viomycin. Supported by NIH grant GM080376 to YEG and BSC and AHA fellowship 12POST8910014 to CC.

In Escherichia coli, 4.5S RNA is found in complexes with both protein translocation protein, Ffh (a bacterial homolog of mammalian SRP54) and protein synthesis elongation factor G (EF-G). To analyze the function of 4.5S RNA in translation, we initially assessed the sensitivity of the association of 4.5S RNA with the ribosome after treatment with antibiotics that affect various stages of protein synthesis. Fusidic acid and viomycin caused 4.5S RNA to cosediment with the 70S ribosomal fraction, indicating that 4.5S RNA enters the ribosome before ribosomal translocation and release of EF-G–GDP from the ribosome. On the other hand, depletion of 4.5S RNA led to the retention of a significant amount of EF-G on 70S ribosomes. In addition, 4.5S RNA shares a conserved decanucleotide sequence (58GAAGCAGCCA67) motif with the characterized EF-G-binding site at positions 1068–1077 on 23S RNA. We therefore examined by gel mobility-shift assay whether or not mutations in the domain-IV region of 4.5S RNA, including this conserved motif, disturb the binding of EF-G to 23S RNA. Any mutation at the C62, G64 or A67 residues within this motif abolished competition activity. Therefore, we propose that 4.5S RNA is concerned with the mode of association of EF-G with the ribosomes. Moreover, this function depends on the secondary structure of 4.5S RNA as well as a ten-base sequence conserved between the two RNAs.

AnEscherichia colimutant lacking the modified nucleotide m1G in rRNA has previously been isolated (G. R. Björk and L. A. Isaksson, J. Mol. Biol. 51:83–100, 1970). In this study, we localize the position of the m1G to nucleotide 745 in 23S rRNA and characterize a mutant deficient in this modification. This mutant shows a 40% decreased growth rate in rich media, a drastic reduction in loosely coupled ribosomes, a 20% decreased polypeptide chain elongation rate, and increased resistance to the ribosome binding antibiotic viomycin. TherrmAgene encoding 23S rRNA m1G745 methyltransferase was mapped to bp 1904000 on theE. colichromosome and identified to be identical to the previously sequenced geneyebH.

Peptidyl-transfer RNA normally dissociates at a low rate from the ribosomes of Escherichia coli during protein synthesis but accumulates under nonpermissive conditions in cells with a temperature-sensitive allele (pth ts ) of the gene encoding peptidyl-transfer RNA hydrolase. The antibiotic-hypersensitive strain E. coli DB-11 with the pth tS mutation was exposed to viomycin, then placed at nonpermissive temperatures. Under these conditions in the absence of drugs, peptidyl-tRNA accumulates, protein synthesis is inhibited and pth ts cells die. When viomycin was present at sufficient concentration to arrest protein synthesis, cell death was not accelerated, error-inducing effects of streptomycin were not counteracted and, at high doses, cytoplasmic accumulation of peptidyl-transfer RNA was slowed down. Blocking the translocation of peptidyl-transfer RNA with viomycin did not stimulate its dissociation from ribosomes. Erythromycin-enhanced cell death was not affected by viomycin at doses sufficient to block amino acid incorporation, suggesting that short peptidyl-transfer RNAs could still be synthesized and dissociated from ribosomes.

Translocation is the step in translation where the peptidyl A-site tRNA on the ribosome moves to the P site and the deacylated P-site tRNA moves to the E site. Recently, several single-molecule FRET studies and cryo-EM studies have confirmed the existence of the tRNA hybrid states (A/P and P/E) and the spontaneous ratchet motion of the ribosome from Macrostate I to Macrostate II prior to translocation (Agirrezabala et al., 2008; Ermolenko et al., 2007a,b; J. Fu, J.B. Munro, S. Blanchard, J. Frank, unpublished). In one of the studies, antibiotic viomycin, which is known to block translation, was shown to promote MS II and the hybrid states the tRNA (Ermolenko et al., 2007B). To determine the mechanism by which viomycin blocks translation, and to further understand the intermediate states during translocation, we studied a pre-translocational complex prepared with viomycin. Single-particle reconstruction was used to determine the structure of the complex. Subsequent classification resulted in two distinct ribosome complexes in MS I and II. We believe that MS II represents the state in which translation is stalled by viomycin. Subsequent analysis revealed that the A-site tRNA is in the A/P hybrid state and the P site tRNA in a novel position in which it makes extensive contacts with the L1 stalk. The results confirmed the observation, by the single molecule FRET study, that viomycin locks the ribosome in the hybrid state. We are setting out for a more detailed analysis to understand the molecular details of the viomycin-induced hybrid states.X. Agirrezabala et al., Mol. Cell, in press (2008).D. N. Ermolenko et al., Nat. Struct. Mol. Biol. 14, 493 (2007a)D. N. Ermolenko et al., J Mol Biol. 370, 530 (2007b).J. B. Munro. et al., Mol. Cell. 25, 505 (2007).

Viomycin and capreomycin, antibiotics produced by Streptomyces vinaceus and S. capreolus respectively, are potent inhibitors of bacterial protein synthesis. Although these organisms are highly tolerant of their own products in vivo, their ribosomes are fully sensitive to the action of the drugs in vitro. However, they possess novel, antibiotic-inactivating enzymes (viomycin phosphotransferase, capreomycin phosphotransferase, capreomycin acetyl-transferase) which, in addition to possible biosynthetic roles, may contribute to the resistances observed in vivo.

Viomycin-resistant strains isolated from Mycobacterium smegmatis demonstrated pleiotropic resistance to tuberactinomycin- N , capreomycin, streptomycin, and kanamycin as a result of mutational alteration of ribosomes, even though they were selected for resistance to a single antibiotic. The pleiotropic drug resistance of three mutants isolated by stepwise selection for resistance to viomycin was due to alteration of the 30 S ribosomal subunit. One mutant, strain A, isolated independently by multiple-step selection to viomycin resistance, was resistant to viomycin, tuberactinomycin- N , and capreomycin through an alteration of the 50 S ribosomal subunit, whereas it was sensitive to kanamycin but resistant to streptomycin through an alteration of the 30 S ribosomal subunit. Three streptomycin-resistant strains, which were isolated by one-step selection at a high concentration of streptomycin, did not show significant co-resistance to any other antibiotics tested in culture and cell-free systems; streptomycin resistance in these mutants was localized on the 30 S ribosomal subunit.

Kanamycin and viomycin were found to block a single cycle of translocation on the poly[U]-ribosome, carrying N-acetyl-diPhe-tRNA on the acceptor site and deacylated tRNA at the donor site. The inhibition of translocation was demonstrated by enhanced puromycin-reactivity of N-acetyl-diPhe-tRNA and by release of deacylated tRNA. The GTPase reaction, catalyzed by EF-G and ribosomes, was not significantly affected by the antibotics. The results with kanamycin and viomycin differed from those obtained with fusidic acid, indicating that the mechanism of translocation inhibition may be different. Kanamycin and viomycin seemed to interfere with the translocation by fixing N-acetyl-Phe-tRNA to the acceptor site, but not to the donor site.

Two alleles for viomycin-capreomycin resistance ( vic ) in Mycobacterium smegmatis affect ribosome structures. One ( vicA ) affects a component of 50 S subunits and the other ( vicB ) affects a component of 30 S subunits. The locus for neomycin-kanamycin resistance ( nek ), which is linked to vicA and vicB , affects a component of 30 S subunits. Although the erythromycin resistance locus ( ery ) is linked to vic and nek , no ribosomal alterations could be detected. Mutations at the streptomycin locus ( str ) not linked to vic and nek caused alterations of 30 S subunits.

The biochemical mechanism of resistance to kanamycin, viomycin, and rifampin in five clinical isolates of Mycobacterium tuberculosis was studied. Resistance to viomycin and kanamycin was attributed to altered ribosomes, whereas resistance to rifampin was attributed to an alteration of RNA polymerase. Ribosomal resistance was, however, not the only way of expressing resistance to viomycin and kanamycin.

The binding of 14C-labelled tuberactinomycin O was analysed in equilibrium dialysis cells. The ionic conditions and the concentration of the labelled drug used in the binding assays allowed the binding of just one drug molecule per non-programmed ribosome. Under these conditions, the occupation of the ribosomal P-site by deacylated tRNAPhe in the presence of poly(U) increased the amount of [14C]tuberactinomycin O bound by a factor of two. Kanamycin, gentamicin and neomycin reduced the binding of tuberactinomycin O, whereas chloramphenicol, tetracycline, streptomycin and puromycin had no effect. A stimulation of the binding of tuberactinomycin O was found upon addition of erythromycin.

The turnover of complexes containing elongation factor (EF) G, the ribosome and either GDP plus fusidic acid or guanyl-5′-yl methylene diphosphonate or guanyl-5′-yl imidodiphosphate has been measured by chasing the complexed, radioactive forms of these guanosine nucleotides with either an excess of unlabelled guanosine nucleotide or the antibiotic thiostrepton. With the three different complexes, the decay promoted by thiostrepton was substantially faster than that induced by the guanosine nucleotide and, under both decay conditions, no simple first-order kinetics were observed. Assuming that the dissociation of the complexes proceeds via a guanosine-nucleotide · EF-G binary complex and a free ribosome, this result suggests that dissociation was reversible and that after it the inactivation of free ribosomes by thiostrepton was faster than the decay of the newly formed binary complex. Increasing the NH4+ concentration from 10 to 180 mM or decreasing the Mg2+ concentration from 30 to 10 mM during measurement of turnover accelerated the chase of [3H]GDP from a [3H]GDP · EF-G · ribosome · fusidic-acid complex. Since these variations did not affect the amount of complex present in solution, they evidently accelerated the reactions leading to formation of the complex. In contrast, increasing the NH4+ concentration of the wash buffer used to isolate the complexes on nitrocellulose membranes sharply decreased their recovery. The antibiotics tetracycline, viomycin and several aminoglycosides that induce misreading (streptomycin, neomycin, kanamycin, tobramycin, gentamicin and paromomycin) inhibited the decay of the GDP · EF-G · ribosome · fusidic acid complex, but other aminoglycosides like spectinomycin and kasugamycin had no effect. Several peptidyl transferase inhibitors (chloramphenicol, lincomycin, viridogrisein, griseoviridin, spiramycin, gougerotin and althiomycin), the EF-Tu-inactivating kirromycin and other antibiotics of less well defined mode of action (erythromycin, bottromycin and micrococcin) were also ineffective. Streptomycin and neomycin were effective at concentrations lower than 10 μM thus indicating that they were probably acting specifically and not as a result of a Mg2+ -like action caused by their polycationic nature. The antibiotics did not completely block turnover and their effect could be explained by an increase in the affinity of the ribosome for the binary GDP · EF-G complex. Since all the effective antibiotics bind to the 30-S subunit and affect functions related to the ribosomal A-site, the results emphasize the important role of this subunit for EF-G interaction and suggest that EF-G binds to the ribosome in a region near the 30-S portion of the A-site. A ternary complex containing Phe-tRNA, EF-Tu and GTP did not modify the chase rate of [14C]GDP from a [14C]GDP · EF-G · ribosome · poly(uridylic acid) · fusidic-acid complex.

The requirements for the decoding process at the ribosomal A site have been investigated in the presence of viomycin. For these studies natural mRNA was replaced either by the synthetic oligonucleotide A-U-G(-U)n, with 0 less than or equal to n less than or equal to 4, or by a physical mixture of the oligonucleotides A-U-G and various oligo(U) sequences. Thus the effect of the "removal" of selected covalent bonds from the sequence A-U-G(U)n could be studied. When the ribosomal P site contains tRNAMetf, then normally the full hexanucleotide "messenger" A-U-G-U-U-U is needed for the EF-Tu-mediated binding of Phe-tRNA into the A site. However in presence of viomycin the pentanucleotide A-U-G-U-U suffices for this. It is also possible in the presence of viomycin to replace A-U-G-U and U-U. In all the above systems the binding of Phe-tRNA required the presence of EF-Tu and GTP. The results suggest that viomycin reinforces interactions between aa-tRNA and the A site after the codon-anticodon recognition step.

The degrees of binding of [3H]dibekacin to LiCl-treated cores of E. coli ribosomes were reduced by increasing LiCl concentrations. The 1.15 M LiCl core lost 70 approximately 80% of the original binding capacity. The antibiotic attachment to the 1.15 M LiCl core was restored by reconstitution with the split proteins (SP), which were obtained by the treatment of 70S ribosomes with LiCl at concentrations of 0.8 approximately 1.15 M. The basic proteins, split off during the transition from 0.4 M LiCl core to 0.8 approximately 1.15 M LiCl core, seemed to be involved in the drug binding. SP0.4 approximately 1.15, which was obtained by the treatment of the 0.4 M LiCl core with 1.15 M LiCl, was fractionated by CM-Sephadex C-25 column chromatography, and each fraction was assayed for protein composition and the capability of restoring the ability of the 1.15 M LiCl core to bind the drug. Of ribosomal proteins eliminated with 1.15 M LiCl, the addition of either S9 or L6 alone to the 1.15 M LiCl core was observed to restore approximately 50% of the binding as compared to the 70S ribosome alone, and both proteins restored about 70% of the binding. The results suggested that ribosomal proteins S9 and L6 were involved in the attachment of [3H]dibekacin to the ribosome. The antibiotic binding to the 70S ribosome and 1.15 M LiCl core reconstituted with S9 and L6 was considerably inhibited by unlabelled dibekacin or kanamycin, and partially inhibited by gentamicin or neomycin, but was not significantly affected by streptomycin or viomycin.

The effect of about 20 aminoglycoside antibiotics comprising compounds with specific 70S or with 70S plus 80S activity on polypeptide synthesis and translational misreading by ribosomes from the archaebacterium Methanococcus vannielii was investigated. A clear structure-activity relationship was found: sensitivity was observed only to the class of 4,5-disubstituted deoxystreptamine compounds, with neomycin and paromomycin as the most active ones. The streptomycin class aminoglycosides were completely inactive whereas the gentamicin group compounds solely affected misreading and only at high concentrations. Viomycin, a specific inhibitor of the translocation reaction at the eubacterial ribosome which competes with binding of 2-deoxystreptamine aminoglycosides was inactive as well.

Binding studies were performed with a [14C]-labelled derivative of viomycin, tuberactinomycin 0 (TUM O). TUM O bound to 30S and 50S subunits. The binding component was the RNA, since ribosomal proteins did not bind the drug. Other RNAs such as tRNA, phage RNA (MS2), and homopolynucleotides also bound the drug. Striking differences in the binding capacity of the various homopolynucleotides were found. Poly(U) bound strongly, poly(G) and poly(C) bound intermediately, whereas poly(A) showed a very low binding. DNA also bound TUM O, although with native DNA the binding was only weak. Finally the effects of viomycin on the assembly in vitro of the 50S subunit from E. coli were tested. A very strong inhibition was found: when the reconstitution was performed at 0.5 x 10(-6) M viomycin the particles formed sedimented at about 50S, but showed a residual activity of less than 10%. The inhibitory power of viomycin with respect to the in vitro assembly is more pronounced than that found in in vitro systems for protein synthesis.

Using dimethyl sulfate and kethoxal, we have probed antibiotic-ribosome complexes, and identified sites of interaction of chloramphenicol, erythromycin, carbomycin, vernamycin B and viomycin with 23S rRNA. Chloramphenicol, erythromycin, carbomycin and vernamycin B protect overlapping nonequivalent sites in the central loop of domain V. From the known functional effects of these drugs and their protection patterns, we infer that peptidyl transferase is inhibited as a result of binding antibiotics proximal to A-2451, whereas antibiotics bound proximal to A-2058 interfere with growth of the nascent polypeptide chain. Vernamycin B also strongly protects A-752, implying that this region of domain II is proximal to the central loop of domain V. Viomycin, which affects translocation and subunit dissociation, protects U-913 and G-914.