Characterization of GE82832, a peptide inhibitor of translocation interacting with bacterial 30S ribosomal subunits

GE82832, a secondary metabolite produced by Streptosporangium cinnabarinum (strain GE82832), has been identified as a translational inhibitor by in vitro screening of a library of natural products. Secondary functional tests specific for individual steps of the translational pathway demonstrated that translocation is the specific target of GE82832. Chemical probing in situ demonstrated that this antibiotic protects bases A1324 and A1333 and exposes C1336 of 16S rRNA, thereby indicating that its binding site is located on the head of the 30S ribosomal subunit. The ribosomal location of GE82832, near ribosomal protein S13 and G1338, two elements of the small subunit that are part of or close to the B1a intrasubunit bridge, suggests that translocation inhibition results from an altered dynamics of 30S-50S ribosomal subunit interaction.     

Comparing FRET Pairs that Report on Intersubunit Rotation in Bacterial Ribosomes

Mediated by elongation factor G (EF-G), ribosome translocation along mRNA is accompanied by rotational movement between ribosomal subunits. Here, we reassess whether the intersubunit rotation requires GTP hydrolysis by EF-G or can occur spontaneously. To that end, we employ two independent FRET assays, which are based on labeling either ribosomal proteins (bS6 and bL9) or rRNAs (h44 of 16S and H101 of 23S rRNA). Both FRET pairs reveal three FRET states, corresponding to the non-rotated, rotated and semi-rotated conformations of the ribosome. Both FRET assays show that in the absence of EF-G, pre-translocation ribosomes containing deacylated P-site tRNA undergo spontaneous intersubunit rotations between non-rotated and rotated conformations. While the two FRET pairs exhibit largely similar behavior, they substantially differ in the fraction of ribosomes showing spontaneous fluctuations. Nevertheless, instead of being an invariable intrinsic property of each FRET pair, the fraction of spontaneously fluctuating molecules changes in both FRET assays depending on experimental conditions. Our results underscore importance of using multiple FRET pairs in studies of ribosome dynamics and highlight the role of thermally-driven large-scale ribosome rearrangements in translation.     

Contribution of intersubunit bridges to the energy barrier of ribosomal translocation

In every round of translation elongation, EF-G catalyzes translocation, the movement of tRNAs (and paired codons) to their adjacent binding sites in the ribosome. Previous kinetic studies have shown that the rate of tRNA–mRNA movement is limited by a conformational change in the ribosome termed ‘unlocking’. Although structural studies offer some clues as to what unlocking might entail, the molecular basis of this conformational change remains an open question. In this study, the contribution of intersubunit bridges to the energy barrier of translocation was systematically investigated. Unlike those targeting B2a and B3, mutations that disrupt bridges B1a, B4, B7a and B8 increased the maximal rate of both forward (EF-G dependent) and reverse (spontaneous) translocation. As bridge B1a is predicted to constrain 30S head movement and B4, B7a and B8 are predicted to constrain intersubunit rotation, these data provide evidence that formation of the unlocked (transition) state involves both 30S head movement and intersubunit rotation.     

Structural dynamics of translating ribosomes.

We describe three groups of small angle neutron scattering (SANS) experiments with translating ribosomes: 1) regular protonated (normal abundance hydrogen) particles; 2) two isotopic hybrid particles which are reconstituted from one protonated and the other deuterated subunit; 3) four isotypic hybrid particles differing from each other by the extent of protein and RNA deuteration. Using the SANS contrast variation method the radii of gyration of protein and RNA components in both ribosomal subunits as well as the intersubunit distance in the pre- and post-translocation states were determined. The results obtained suggest the following model of the ribosome as a dynamic machine. The ribosome oscillates between two major conformers differing in geometrical dimensions. The 'active' (pulsating) part of the ribosome is the 30S subunit. We believe that the movement of its 'head' relative to the passive 50S subunit is the main mechanical act of translocation. The radius of gyration of the 30S subunit and the intersubunit distance change upon the movement. This is corroborated by neutron scattering data.     

Observation of intersubunit movement of the ribosome in solution using FRET

Protein synthesis is believed to be a dynamic process, involving structural rearrangements of the ribosome. Cryo-EM reconstructions of certain elongation factor G (EF-G)-containing complexes have led to the proposal that translocation of tRNA and mRNA through the ribosome, from the A to P to E sites, is accompanied by a rotational movement between the two ribosomal subunits. Here, we have used F?rster resonance energy transfer (FRET) to monitor changes in the relative orientation of the ribosomal subunits in different complexes trapped at intermediate stages of translocation in solution. Binding of EF-G to the ribosome in the presence of the non-hydrolyzable GTP analogue GDPNP or GTP plus fusidic acid causes an increase in the efficiency of energy transfer between fluorophores introduced into proteins S11 in the 30 S subunit and L9 in the 50 S subunit, and a decrease in energy transfer between S6 and L9. Similar anti-correlated changes in energy transfer occur upon binding the GTP-requiring release factor RF3. These changes are consistent with the counter-clockwise rotation of the 30 S subunit relative to the 50 S subunit observed in cryo-EM studies. Reaction of ribosomal complexes containing the peptidyl-tRNA analogues N-Ac-Phe-tRNAPhe, N-Ac-Met-tRNAMet or f-Met-tRNAfMet with puromycin, conditions favoring movement of the resulting deacylated tRNAs into the P/E hybrid state, leads to similar changes in FRET. Conversely, treatment of a ribosomal complex containing deacylated and peptidyl-tRNAs bound in the A/P and P/E states, respectively, with EF-G.GTP causes reversal of the FRET changes. The use of FRET has enabled direct observation of intersubunit movement in solution, provides independent evidence that formation of the hybrid state is coupled to rotation of the 30 S subunit and shows that the intersubunit movement is reversed during the second step of translocation.     

Spontaneous intersubunit rotation in single ribosomes

During the elongation cycle, tRNA and mRNA undergo coupled translocation through the ribosome catalyzed by elongation factor G (EF-G). Cryo-EM reconstructions of certain EF-G-containing complexes led to the proposal that the mechanism of translocation involves rotational movement between the two ribosomal subunits. Here, using single-molecule FRET, we observe that pretranslocation ribosomes undergo spontaneous intersubunit rotational movement in the absence of EF-G, fluctuating between two conformations corresponding to the classical and hybrid states of the translocational cycle. In contrast, posttranslocation ribosomes are fixed predominantly in the classical, nonrotated state. Movement of the acceptor stem of deacylated tRNA into the 50S E site and EF-G binding to the ribosome both contribute to stabilization of the rotated, hybrid state. Furthermore, the acylation state of P site tRNA has a dramatic effect on the frequency of intersubunit rotation. Our results provide direct evidence that the intersubunit rotation that underlies ribosomal translocation is thermally driven.     

New insights into the enzymatic role of EF-G in ribosome recycling

During translation, elongation factor G (EF-G) plays a catalytic role in tRNA translocation and a facilitative role in ribosome recycling. By stabilizing the rotated ribosome and interacting with ribosome recycling factor (RRF), EF-G was hypothesized to induce the domain rotations of RRF, which subsequently performs the function of splitting the major intersubunit bridges and thus separates the ribosome into subunits for recycling. Here, with systematic mutagenesis, FRET analysis and cryo-EM single particle approach, we analyzed the interplay between EF-G/RRF and post termination complex (PoTC). Our data reveal that the two conserved loops (loop I and II) at the tip region of EF-G domain IV possess distinct roles in tRNA translocation and ribosome recycling. Specifically, loop II might be directly involved in disrupting the main intersubunit bridge B2a between helix 44 (h44 from the 30S subunit) and helix 69 (H69 from the 50S subunit) in PoTC. Therefore, our data suggest a new ribosome recycling mechanism which requires an active involvement of EF-G. In addition to supporting RRF, EF-G plays an enzymatic role in destabilizing B2a via its loop II.     

Localization of the protein L2 in the 50 S subunit and the 70 S E. coli ribosome

The protein L2 is found in all ribosomes and is one of the best conserved proteins of this mega-dalton complex. The protein was localized within both the isolated 50 S subunit and the 70 S ribosome of the Escherichia coli bacteria with the neutron-scattering technique of spin-contrast variation. L2 is elongated, exposing one end of the protein to the surface of the intersubunit interface of the 50 S subunit. The protein changes its conformation slightly when the 50 S subunit reassociates with the 30 S subunit to form a 70 S ribosome, becoming more elongated and moving approximately 30 A into the 50 S matrix. The results support a recent observation that L2 is essential for the association of the ribosomal subunits and might participate in the binding and translocation of the tRNAs.     

Mutations in the intersubunit bridge regions of 23 S rRNA

The large and small subunits of the ribosome are joined by a series of bridges that are conserved among mitochondrial, bacterial, and eukaryal ribosomes. In addition to joining the subunits together at the initiation of protein synthesis, a variety of other roles have been proposed for these bridges. These roles include transmission of signals between the functional centers of the two subunits, modulation of tRNA-ribosome and factor-ribosome interactions, and mediation of the relative movement of large and small ribosomal subunits during translocation. The majority of the bridges involve RNA-RNA interactions, and to gain insight into their function, we constructed mutations in the 23 S rRNA regions involved in forming 7 of the 12 intersubunit bridges in the Escherichia coli ribosome. The majority of the mutants were viable in strains expressing mutant rRNA exclusively but had distinct growth phenotypes, particularly at 30 degrees C, and the mutant ribosomes promoted a variety of miscoding errors. Analysis of subunit association activities both in vitro and in vivo indicated that, with the exception of the bridge B5 mutants, at least one mutation at each bridge site affected 70 S ribosome formation. These results confirm the structural data linking bridges with subunit-subunit interactions and, together with the effects on decoding fidelity, indicate that intersubunit bridges function at multiple stages of protein synthesis.     

Molecular dynamics of EF-G during translocation

Elongation factor G (EF‐G) plays a crucial role in two stages of mRNA‐(tRNA)₂ translocation. First, EF‐G?GTP enters the pre‐translocational ribosome in its intersubunit‐rotated state, with tRNAs in their hybrid (P/E and A/P) positions. Second, a conformational change in EF‐G's Domain IV induced by GTP hydrolysis disengages the mRNA‐anticodon stem‐loops of the tRNAs from the decoding center to advance relative to the small subunit when the ribosome undergoes a backward inter‐subunit rotation. These events take place as EF‐G undergoes a series of large conformational changes as visualized by cryo‐EM and X‐ray studies. The number and variety of these structures leave open many questions on how these different configurations form during the dynamic translocation process. To understand the molecular mechanism of translocation, we examined the molecular motions of EF‐G in solution by means of molecular dynamics simulations. Our results show: (1) rotations of the super‐domain formed by Domains III–V with respect to the super‐domain formed by I–II, and rotations of Domain IV with respect to Domain III; (2) flexible conformations of both 503‐ and 575‐loops; (3) large conformational variability in the bound form caused by the interaction between Domain V and the GTPase‐associated center; (4) after GTP hydrolysis, the Switch I region seems to be instrumental for effecting the conformational change at the end of Domain IV implicated in the disengagement of the codon‐anticodon helix from the decoding center. Proteins 2011; © 2011 Wiley‐Liss, Inc.     

Dynamic contact network between ribosomal subunits enables rapid large-scale rotation during spontaneous translocation

During ribosomal translation, the two ribosomal subunits remain associated through intersubunit bridges, despite rapid large-scale intersubunit rotation. The absence of large barriers hindering rotation is a prerequisite for rapid rotation. Here, we investigate how such a flat free-energy landscape is achieved, in particular considering the large shifts the bridges undergo at the periphery. The dynamics and energetics of the intersubunit contact network are studied using molecular dynamics simulations of the prokaryotic ribosome in intermediate states of spontaneous translocation. Based on observed occupancies of intersubunit contacts, residues were grouped into clusters. In addition to the central contact clusters, peripheral clusters were found to maintain strong steady interactions by changing contacts in the course of rotation. The peripheral B1 bridges are stabilized by a changing contact pattern of charged residues that adapts to the rotational state. In contrast, steady strong interactions of the B4 bridge are ensured by the flexible helix H34 following the movement of protein S15. The tRNAs which span the subunits contribute to the intersubunit binding enthalpy to an almost constant degree, despite their different positions in the ribosome. These mechanisms keep the intersubunit interaction strong and steady during rotation, thereby preventing dissociation and enabling rapid rotation.     

Structural insights into initial and intermediate steps of the ribosome-recycling process

The ribosome-recycling factor (RRF) and elongation factor-G (EF-G) disassemble the 70S post-termination complex (PoTC) into mRNA, tRNA, and two ribosomal subunits. We have determined cryo-electron microscopic structures of the PoTC·RRF complex, with and without EF-G. We find that domain II of RRF initially interacts with universally conserved residues of the 23S rRNA helices 43 and 95, and protein L11 within the 50S ribosomal subunit. Upon EF-G binding, both RRF and tRNA are driven towards the tRNA-exit (E) site, with a large rotational movement of domain II of RRF towards the 30S ribosomal subunit. During this intermediate step of the recycling process, domain II of RRF and domain IV of EF-G adopt hitherto unknown conformations. Furthermore, binding of EF-G to the PoTC·RRF complex reverts the ribosome from ratcheted to unratcheted state. These results suggest that (i) the ribosomal intersubunit reorganizations upon RRF binding and subsequent EF-G binding could be instrumental in destabilizing the PoTC and (ii) the modes of action of EF-G during tRNA translocation and ribosome-recycling steps are markedly different.     

Analysis of the function of <Emphasis Type="Italic">E. coli</Emphasis> 23S rRNA helix-loop 69 by mutagenesis

Background  

The ribosome is a two-subunit enzyme known to exhibit structural dynamism during protein synthesis. The intersubunit bridges have been proposed to play important roles in decoding, translocation, and the peptidyl transferase reaction; yet the physical nature of their contributions is ill understood. An intriguing intersubunit bridge, B2a, which contains 23S rRNA helix 69 as a major component, has been implicated by proximity in a number of catalytically important regions. In addition to contacting the small ribosomal subunit, helix 69 contacts both the A and P site tRNAs and several translation factors.     

A thermal ratchet model of tRNA–mRNA translocation by the ribosome

The translocation of tRNA coupled with mRNA in the ribosome is one of important steps during protein synthesis. Despite extensive experimental studies, the detailed mechanism of the translocation remains undetermined. Here, based on previous biochemical, cryo-electron microscopic and X-ray crystallographic studies, a thermal ratchet model is presented for this translocation. In the model, during one elongation cycle of the protein synthesis, two large conformational transitions of the ribosome are involved, with one being the relative rotation between the two ribosomal subunits following the peptide transfer, which is facilitated by the EF-G.GTP binding, and the other one being the reverse relative rotation between the two ribosomal subunits upon EF-G.GTP hydrolysis. The former conformational change plays an important role in ensuring the completion of the release of the deacylated tRNA from the ribosome before tRNA–mRNA translocation. The latter reverse conformational change upon GTP hydrolysis is followed by rapid tRNA–mRNA translocation and Pi release, both of which take place independently of each other. This is consistent with the previous biochemical experimental data. Also, the model is consistent with other available experimental results such as the suppression of EF-G-dependent translocation in cross-linked ribosomes and frameshifting under some conditions.     

Structural basis for the enhanced thermal stability of alcohol dehydrogenase mutants from the mesophilic bacterium Clostridium beijerinckii: contribution of salt bridging

Previous research in our laboratory comparing the three-dimensional structural elements of two highly homologous alcohol dehydrogenases, one from the mesophile Clostridium beijerinckii (CbADH) and the other from the extreme thermophile Thermoanaerobacter brockii (TbADH), suggested that in the thermophilic enzyme, an extra intrasubunit ion pair (Glu224-Lys254) and a short ion-pair network (Lys257-Asp237-Arg304-Glu165) at the intersubunit interface might contribute to the extreme thermal stability of TbADH. In the present study, we used site-directed mutagenesis to replace these structurally strategic residues in CbADH with the corresponding amino acids from TbADH, and we determined the effect of such replacements on the thermal stability of CbADH. Mutations in the intrasubunit ion pair region increased thermostability in the single mutant S254K- and in the double mutant V224E/S254K-CbADH, but not in the single mutant V224E-CbADH. Both single amino acid replacements, M304R- and Q165E-CbADH, in the region of the intersubunit ion pair network augmented thermal stability, with an additive effect in the double mutant M304R/Q165E-CbADH. To investigate the precise mechanism by which such mutations alter the molecular structure of CbADH to achieve enhanced thermostability, we constructed a quadruple mutant V224E/S254K/Q165E/M304R-CbADH and solved its three-dimensional structure. The overall results indicate that the amino acid substitutions in CbADH mutants with enhanced thermal stability reinforce the quaternary structure of the enzyme by formation of an extended network of intersubunit ion pairs and salt bridges, mediated by water molecules, and by forming a new intrasubunit salt bridge.     

Multiple effects of S13 in modulating the strength of intersubunit interactions in the ribosome during translation

The ribosomal protein S13 is found in the head region of the small subunit, where it interacts with the central protuberance of the large ribosomal subunit and with the P site-bound tRNA through its extended C terminus. The bridging interactions between the large and small subunits are dynamic, and are thought to be critical in orchestrating the molecular motions of the translation cycle. S13 provides a direct link between the tRNA-binding site and the movements in the head of the small subunit seen during translocation, thereby providing a possible pathway of signal transduction. We have created and characterized an rpsM(S13)-deficient strain of Escherichia coli and have found significant defects in subunit association, initiation and translocation through in vitro assays of S13-deficient ribosomes. Targeted mutagenesis of specific bridge and tRNA contact elements in S13 provides evidence that these two interaction domains play critical roles in maintaining the fidelity of translation. This ribosomal protein thus appears to play a non-essential, yet important role by modulating subunit interactions in multiple steps of the translation cycle.     

Site-directed fluorescence labeling reveals differences on the R-conformer of glucosamine 6-phosphate deaminase of Escherichia coli induced by active or allosteric site ligands at steady state

Engineered glucosamine 6-phosphate deaminase of Escherichia coli with unique reactive cysteines at positions 164 or 206 was created by site-directed mutagenesis to monitor the allosteric transition in solution by the fluorescence emission of the bimane or dansyl-amidoethyl groups attached to the indicated residues. The selection of both positions was due to the differential interaction of these residues between T- and R-conformers at the interface of two trimers that form the hexameric structure; in the T-conformer, residue 164 or 206 presents only intrasubunit contacts, but in the R-conformer, new intersubunit contacts are established. As in the wild-type enzyme, fluorescent-labeled mutants show no modification on the allosteric activation of the K-system, only the kcat was reduced to a value of 72 s(-1) (approximately 50% of wild-type). With these preparations, conformational changes were detected by the fluorescence emission spectra at steady state when the active site or the allosteric site ligands were titrated. Despite the similar changes in the fluorescence spectra that were correlated with the induction of the R-state, differences were observed at the maximal change in the fluorescence spectra and in the relative solvent polarities at the positions labeled. These data suggested structural differences in the conformation of the R-state when it is induced from the active site or from the allosteric site, which is not consistent with the two-state structural model proposed by previous crystallographic studies of this enzyme.     

Structural basis for hygromycin B inhibition of protein biosynthesis

Aminoglycosides are one of the most widely used and clinically important classes of antibiotics that target the ribosome. Hygromycin B is an atypical aminoglycoside antibiotic with unique structural and functional properties. Here we describe the structure of the intact Escherichia coli 70S ribosome in complex with hygromycin B. The antibiotic binds to the mRNA decoding center in the small (30S) ribosomal subunit of the 70S ribosome and induces a localized conformational change, in contrast to its effects observed in the structure of the isolated 30S ribosomal subunit in complex with the drug. The conformational change in the ribosome caused by hygromycin B binding differs from that induced by other aminoglycosides. Also, in contrast to other aminoglycosides, hygromycin B potently inhibits spontaneous reverse translocation of tRNAs and mRNA on the ribosome in vitro. These structural and biochemical results help to explain the unique mode of translation inhibition by hygromycin B.