Empty Ribosomes Research Articles

A binding site for the antibiotic GE81112 in the ribosomal mRNA channel.

The initiation phase is the rate-limiting step of protein synthesis (translation) and is finely regulated, making it an important drug target. In bacteria, initiation is guided by three initiation factors and involves positioning the start site on the messenger RNA within the P-site on the small ribosomal subunit (30S), where it is decoded by the initiator tRNA. This process can be efficiently inhibited by GE81112, a natural hydrophilic, noncyclic, nonribosomal tetrapeptide. It is found in nature in three structural variants (A, B and B1 with molecular masses of 643-658 Da). Previous biochemical and structural characterisation of GE81112 indicates that the primary mechanism of action of this antibiotic is to (1) prevent the initiator tRNA from binding correctly to the P-site and (2) block conformational rearrangements in initiation factor IF3, resulting in an unlocked 30S pre IC state. In this study, using cryoEM, we have determined the binding site of GE81112 in initiation complexes (3.2-3.7Å) and on empty ribosomes (2.09 Å). This binding site is within the mRNA channel (E-site) but remote from the binding site of the initiation factors and initiator tRNA. This suggests that it acts allosterically to prevent the initiator tRNA from being locked into place. The binding mode is consistent with previous biochemical studies and recent work identifying the key pharmacophores of GE81112.

Nascent-chain interaction networks and their effect on the bacterial ribosome.

In order to become bioactive, proteins need to be biosynthesized and protected from aggregation during translation. Remarkably, the ribosome contributes to both of these tasks. Some ribosomal proteins (r-proteins) are known to noncovalently interact with ribosome-bound nascent chains (RNCs). Yet, specific interaction networks and their role within the ribosomal machinery remain poorly characterized. Here, we found that empty ribosomes are more sensitive to urea denaturation than ribosomes harboring nascent protein chains, including aminoacyl or short peptidyl-tRNA (snc-tRNA). This result suggests that snc-tRNAs play a ribosome-stabilizing role. On the other hand, RNCs of variable sequence and length do not significantly modify the apparent stability of the peptidyl-transferase center (PTC) and r-proteins, suggesting that RNC/r-protein interaction networks close to the PTC have a negligible effect on the apparent ribosome stability. Fluorescence anisotropy decay, chemical-crosslinking and Western blots show that 140-residue-long RNCs of the foldable protein apoHmp have an N-terminal compact region (63-94 residues) and interact with r-protein L23 but not with L24 or L29. Longer RNCs bear a similar compact region and interact either with L23 or with L23 and another unidentified r-protein or with molecular chaperones. Interestingly, the apparent stability of RNC/r-protein complexes does not depend on RNC sequence or length. Taken together, our findings show that RNCs establish preferential interactions with the L23 ribosomal protein whose strength does not depend on RNC amino-acid sequence and length. As the nascent chain elongates, the interaction network extends to additional r-proteins and chaperones. It follows that the ribosome provides uniform, indiscriminate support to nascent proteins regardless of their sequence and foldability. This result highlights the mode of action of this unbiased molecular machine for cellular health.

Contribution of Nascent Polypeptides of increasing Length to the Apparent Stability of the Bacterial Ribosome

The role of the ribosome in the cell extends well beyond catalysis of peptide-bond formation. For instance, the ribosome is emerging as a key player in cotranslational protein folding. On the other hand, whether and how the ribosome assists nascent-protein folding, and how the nascent protein may in turn affect the ribosome, is poorly understood. Recent results suggest that the ribosome facilitatesprotein folding by directly interacting with nascent chains. Yet, whether interactions between the ribosome and the nascent chain have any influence on the ribosome, and whether these interactions promote proper protein folding is currently unknown. Via sucrose-gradient assays, we found that empty ribosomes are more sensitive to urea denaturation than ribosomal complexes carrying either aminoacyl-tRNA or nascent-chains. Further, by taking advantage of tryptophan fluorescence emission, we probed the effect of nascent chains of widely variable sequence and length on the apparent stability of ribosomal proteins (rProteins). We found that there is no statistically significant difference between the contribution of different nascent chains, suggesting that nascent-chain-to-rProtein interactions are either weak or thermodynamically compensated. Hence, there is no difference between the sensitivity to urea of ribosomal initiation complexes carrying aminoacyl tRNA and ribosomal complexes harboring peptidyl tRNA. in all, the above findings lead us to conclude that aminoacyl tRNA, likely in conjunction with mRNA, increases the apparent stability of the ribosome, while the nascent polypeptide does not. Finally, our data led us to propose a multi-step model for the disassembly of ribosome-nascent-chain complexes.

Effect of Nascent Proteins on the Stability of the Ribosome

Recent studies showed that many nascent proteins begin folding during translation, sometimes within the vestibule of the ribosomal exit tunnel. On the other hand, how and whether the ribosome assists protein folding, and how protein folding may in turn affect the ribosome, is poorly understood. We recently detected interactions between foldable and intrinsically disordered nascent proteins (not bearing an N-terminal signal sequence) and ribosomal proteins by dynamic fluorescence depolarization in the frequency domain and by chemical cross-linking. These results suggest that the ribosome facilitates in vivo protein folding by directly interacting with nascent chains. Whether interactions between the ribosome and the nascent chain have any thermodynamic influence on the ribosome, and whether these interactions promote proper protein folding however, is not known. Via sucrose-gradient assays, we found that empty ribosomes are less stable than ribosomes carrying tRNA, or ribosomes harboring nascent-chains. In addition, by taking advantage of tryptophan fluorescence emission, we probed the effect of several nascent chains of variable sequence and length on the stability of ribosomal proteins. We found that there is no statistically significant difference between the contribution of different nascent-chain constructs, suggesting that nascent-to-ribosomal protein interactions are either weak or thermodynamically compensated. Hence, there is no apparent difference between the overall stability of ribosomes carrying a tRNA and ribosomes harboring a nascent-chain. In all, the above findings lead us to conclude that tRNA stabilizes the ribosome, while nascent proteins do not. Therefore, intriguingly, the interplay between the ribosome and nascent chains does not critically rely on standard-state net free-energy changes. Finally, our data led us to propose a multi-step model for the disassembly of ribosome-nascent-chain complexes.

Voltage Sensitivity of the Bacterial Protein Translocation Channel

The heterotrimeric protein translocation channel SecYEG enables (i) soluble proteins to cross the inner membrane and (ii) hydrophobic proteins to enter the membrane interior. It contains an aqueous pore that, in its resting state, is sealed by a ring of six hydrophobic residues and a half helix, termed the plug [1]. Signal sequence binding or ribosome binding both dislocate the plug and break the ring, thereby opening the channel to ions [2]. The membrane barrier to ions is preserved, since physiological values of the transmembrane potential close the channel by a yet unknown mechanism [3]. Here we demonstrate that this voltage sensitivity does not depend on the ligand. To be precise, the open time decreases together with a decrease in voltage for SecYEG channels that are bound to (i) signal peptides, (ii) translocation intermediates (proOmpA) and the motor protein SecA, (iii) a ribosome-nascent chain (FtsQ) complex or (iv) empty ribosomes. The observations were made with planar lipid bilayers that contained the purified and reconstituted SecYEG complex. They indicate that the voltage sensor must be part of the SecYEG channel. In our search for the sensor, we mutated charged residues, deleted the plug and performed various cross-link experiments, the outcome of which will be discussed.1. Saparov, S.M., Erlandson, K., Cannon, K., Schaletzky, J., Schulman, S., Rapoport, T.A., Pohl, P. (2007). Determining the Conductance of the SecY Protein Translocation Channel for Small Molecules. Mol. Cell.2. Knyazev, D.G., Lents, A., Krause, E., Ollinger, N., Siligan, C., Papinski, D., Winter, L., Horner, A., Pohl, P. (2013). The Bacterial Translocon SecYEG Opens upon Ribosome Binding. J. Biol. Chem.3. Knyazev, D.G., Winter, L., Bauer, B.W., Siligan, C., Pohl, P. (2014). Ion Conductivity of the Bacterial Translocation Channel SecYEG Engaged in Translocation. J. Biol. Chem.

Translational control 1995-2015: unveiling molecular underpinnings and roles in human biology.

The last 20 years have seen tremendous progress in deciphering the fundamental mechanisms of protein synthesis and molecular pathways of translational control, and in implicating these processes in development, neurobiology, and etiologyof human diseases. Muchof this success can be attributed to the introduction of new technologies, such as next-generation RNA sequencing and advances in cryo-EM; and also to development of powerful in vitro systems recapitulating complex in vivo phenomena with purified components. Also critical have been strategic interactions between experts in different disciplines of biology, such as the wedding of biochemistry with genetics and structural biology, and of translation biochemistry with neurobiology and medicine. RNA has contributed to this progress by publishing key findings on molecular mechanisms of protein synthesis and the role of translational control in regulating gene expression in diverse organisms. Thebasic reactions of the elongationand termination stages of protein synthesis are highly conserved throughout all three kingdoms of life. Because they occur in the catalytic center of the ribosome, their molecular mechanisms were illuminated by the stunning successes of X-ray crystallography and cryo-electron microscopy in producing high-resolution structures of both empty ribosomes and a myriad of ribosome complexes with mRNA, tRNAs, or elongation, termination, or ribosome recycling factors trapped in different stages of the process. Recent innovations in cryo-EM technology are revealing ribosome structures at nearly 3 A resolution; and new algorithms for sorting structurally distinct molecules means that sample heterogeneity (an anathema to crystallography) is now exploited to identify different conformational states in the same preparation. The solved ribosome structures revealed the path of mRNA and the A, P, and E binding sites for the tRNA, as it progress through the elongation cycle from aminoacyltRNA, to peptidyl-tRNA to deacylated tRNA, located at the interface between the large and small subunits; as well as the peptidyl transferase center wherepeptide bond formation occurs. As the decoding center of the ribosome is comprised almost entirely of rRNA, it became clear that the ribosome is a ribozyme. The protein factors involved in translocation of mRNA and the tRNAs through the decoding sites (EF-G/ EF2), in recognizing stop codons and hydrolyzing peptidyltRNA (RF1/eRF1), and splitting apart ribosomal subunits following termination (bacterial RRF) all exploit striking structural mimicry of tRNA to fit into the A site. Interestingly, RF1 was shown to contain a protein element that “reads” the stop codon and another that reaches into the peptidyl transferase center to hydrolyze the peptidyl-tRNA linkage. The peptidyl-tRNA and aminoacyl-tRNA are anchored in the precise locations required for peptide bond formation, but the ribosome is not simply a rigid catalytic platform, but undergoes conformational changes in selecting the correct EF1A·GTP·aminoacyl-tRNA ternary complex for each codon, and displays inter-subunit ratcheting during translocation and termination. GTP binding and hydrolysis by the elongation factors evoke cyclic conformational changes that drive the process forward. The use of fluorescently labeled reactants and stopped-flow kinetic analysis allowed a detailed dissection of different steps in the processes of aminoacyltRNA selection in the A site, with proofreading of the codon:anticodon interaction; and of translocation of tRNAs through the decoding center catalyzed by EF2. Single-molecule approaches also contributed importantly to this progress, by identifying intermediate states too transient to be detected by ensemble kinetic experiments or structural biology. In other developments, eIF5A and its bacterial counterpart EF-P were shown to stimulate elongation specifically through stretches of proline codons, and both factors occupy the E-site and communicate directly with the peptidyl transferase center. Interestingly, RRF is replaced in eukaryotes and archaea by an ATP-binding cassette protein, ABCE1/ Rli1, that becomes wedged between the large and small subunits and uses ATP hydrolysis to split apart the subunits. ABCE1/Rli1also functions with RF1 paralog Dom34 to

Ribosomes in the balance: structural equilibrium ensures translational fidelity and proper gene expression.

At equilibrium, empty ribosomes freely transit between the rotated and un-rotated states. In the cell, the binding of two translation elongation factors to the same general region of the ribosome stabilizes one state over the other. These stabilized states are resolved by expenditure of energy in the form of GTP hydrolysis. A prior study employing mutants of a late assembling peripheral ribosomal protein suggested that ribosome rotational status determines its affinity for elongation factors, and hence translational fidelity and gene expression. Here, mutants of the early assembling integral ribosomal protein uL2 are used to test the generality of this hypothesis. rRNA structure probing analyses reveal that mutations in the uL2 B7b bridge region shift the equilibrium toward the rotated state, propagating rRNA structural changes to all of the functional centers of ribosome. Structural disequilibrium unbalances ribosome biochemically: rotated ribosomes favor binding of the eEF2 translocase and disfavor that of the elongation ternary complex. This manifests as specific translational fidelity defects, impacting the expression of genes involved in telomere maintenance. A model is presented describing how cyclic intersubunit rotation ensures the unidirectionality of translational elongation, and how perturbation of rotational equilibrium affects specific aspects of translational fidelity and cellular gene expression.

Dynamics of Trigger Factor Interaction with Translating Ribosomes

In all organisms ribosome-associated chaperones assist early steps of protein folding. To elucidate the mechanism of their action, we determined the kinetics of individual steps of the ribosome binding/release cycle of bacterial trigger factor (TF), using fluorescently labeled chaperone and ribosome-nascent chain complexes. Both the association and dissociation rates of TF-ribosome complexes are modulated by nascent chains, whereby their length, sequence, and folding status are influencing parameters. However, the effect of the folding status is modest, indicating that TF can bind small globular domains and accommodate them within its substrate binding cavity. In general, the presence of a nascent chain causes an up to 9-fold increase in the rate of TF association, which provides a kinetic explanation for the observed ability of TF to efficiently compete with other cytosolic chaperones for binding to nascent chains. Furthermore, a subset of longer nascent polypeptides promotes the stabilization of TF-ribosome complexes, which increases the half-life of these complexes from 15 to 50 s. Nascent chains thus regulate their folding environment generated by ribosome-associated chaperones.

Elongation factors in protein synthesis.

Recent discoveries of elongation factor-related proteins have considerably complicated the simple textbook scheme of the peptide chain elongation cycle. During growth and differentiation the cycle may be regulated not only by factor modification but also factor replacement. In addition, rare tRNAs may have their own rare factor proteins. A special case is the acquisition of resistance by bacteria to elongation factor-directed antibiotics. Pertinent data from the literature and our own work with Escherichia coli and Streptomyces are discussed. The GTP-binding domain of EF-Tu has been studied extensively, but little molecular detail is available on the interactions with its other ligands or effectors, or on the way they are affected by the GTPase switch signal. A growing number of EF-Tu mutants obtained by ourselves and others are helping us in testing current ideas. We have found a synergistic effect between EF-Tu and EF-G in their uncoupled GTPase reactions on empty ribosomes. Only the EF-G reaction is perturbed by fluoroaluminates.

Conformational change in the 16S rRNA in the Escherichia coli 70S ribosome induced by P/P- and P/E-site tRNAPhe binding.

The effects of P/P- and P/E-site tRNA(Phe) binding on the 16S rRNA structure in the Escherichia coli 70S ribosome were investigated using UV cross-linking. The identity and frequency of 16S rRNA intramolecular cross-links were determined in the presence of deacyl-tRNA(Phe) or N-acetyl-Phe-tRNA(Phe) using poly(U) or an mRNA analogue containing a single Phe codon. For N-acetyl-Phe-tRNA(Phe) with either poly(U) or the mRNA analogue, the frequency of an intramolecular cross-link C967 x C1400 in the 16S rRNA was decreased in proportion to the binding stoichiometry of the tRNA. A proportional effect was true also for deacyl-tRNA(Phe) with poly(U), but the decrease in the C967 x C1400 frequency was less than the tRNA binding stoichiometry with the mRNA analogue. The inhibition of the C967 x C1400 cross-link was similar in buffers with, or without, polyamines. The exclusive participation of C967 with C1400 in the cross-link was confirmed by RNA sequencing. One intermolecular cross-link, 16S rRNA (C1400) to tRNA(Phe)(U33), was made with either poly(U) or the mRNA analogue. These results indicate a limited structural change in the small subunit around C967 and C1400 during tRNA P-site binding sensitive to the type of mRNA that is used. The absence of the C967 x C1400 cross-link in 70S ribosome complexes with tRNA is consistent with the 30S and 70S crystal structures, which contain tRNA or tRNA analogues; the occurrence of the cross-link indicates an alternative arrangement in this region in empty ribosomes.

Synergism between the GTPase Activities of EF-Tu·GTP and EF-G·GTP on Empty Ribosomes: Elongation Factors as Stimulators of the Ribosomal Oscillation between Two Conformations

A remarkable positive cooperativity between the GTPase activities of EF-Tu and EF-G on empty ribosomes from Escherichia coli has been discovered. This cooperatively implies a decrease of the corresponding apparent KM values of the empty ribosome for either elongation factor: from more than 10 μM to 0·5 μM for EF-Tu·GTP by the addition of 0·25 μM EF-G and from 0·7 μM to 0·5 μM for EF-G·GTP by the addition of 3 μM EF-Tu. In a further analysis of this phenomenon, the effects of various specific antibiotics were studied: thiostrepton, fusidic acid, tetracycline, pulvomycin and kirromycin appeared to inhibit the synergistic effect, whereas streptomycin was found to stimulate it. Even in the present minimal system the ribosomes respond to the above-mentioned antibiotics in a way surprisingly similar to that in the coupled system with mRNA and tRNAs. The cooperativity seems not to be due to a simultaneous binding of the two elongation factors to the ribosome as revealed by studying the effects of fusidic acid and kirromycin, and by band-shift experiments by means of gel electrophoresis under non-denaturing conditions. Our experimental data and the kinetic analysis of alternative models provide evidence that EF-Tu·GTP and EF-G·GTP interact sequentially with empty ribosomes that oscillate between two different conformations, one for each elongation factor. Apparently, ribosomes have an intrinsic property for oscillation as normally observed during protein synthesis with a frequency paced by the events of tRNA binding and translocation.