Localization of L11 protein on the ribosome and elucidation of its involvement in EF-G-dependent translocation

L11 protein is located at the base of the L7/L12 stalk of the 50 S subunit of the Escherichia coli ribosome. Because of the flexible nature of the region, recent X-ray crystallographic studies of the 50 S subunit failed to locate the N-terminal domain of the protein. We have determined the position of the complete L11 protein by comparing a three-dimensional cryo-EM reconstruction of the 70 S ribosome, isolated from a mutant lacking ribosomal protein L11, with the three-dimensional map of the wild-type ribosome. Fitting of the X-ray coordinates of L11-23 S RNA complex and EF-G into the cryo-EM maps combined with molecular modeling, reveals that, following EF-G-dependent GTP hydrolysis, domain V of EF-G intrudes into the cleft between the 23 S ribosomal RNA and the N-terminal domain of L11 (where the antibiotic thiostrepton binds), causing the N-terminal domain to move and thereby inducing the formation of the arc-like connection with the G' domain of EF-G. The results provide a new insight into the mechanism of EF-G-dependent translocation.     

Functional interactions between the G' subdomain of bacterial translation factor EF-G and ribosomal protein L7/L12

Protein L7/L12 of the bacterial ribosome plays an important role in activating the GTP hydrolytic activity of elongation factor G (EF-G), which promotes ribosomal translocation during protein synthesis. Previously, we cross-linked L7/L12 from two residues (209 and 231) flanking alpha-helix AG' in the G' subdomain of Escherichia coli EF-G. Here we report kinetic studies on the functional effects of mutating three neighboring glutamic acid residues (224, 228, and 231) to lysine, either singly or in combination. Two single mutations (E224K and E228K), both within helix AG', caused large defects in GTP hydrolysis and smaller defects in ribosomal translocation. Removal of L7/L12 from the ribosome strongly reduced the activities of wild type EF-G but had no effect on the activities of the E224K and E228K mutants. Together, these results provide evidence for functionally important interactions between helix AG' of EF-G and L7/L12 of the ribosome.     

Translational regulation via L11: molecular switches on the ribosome turned on and off by thiostrepton and micrococcin

The thiopeptide class of antibiotics targets the GTPase-associated center (GAC) of the ribosome to inhibit translation factor function. Using X-ray crystallography, we have determined the binding sites of thiostrepton (Thio), nosiheptide (Nosi), and micrococcin (Micro), on the Deinococcus radiodurans large ribosomal subunit. The thiopeptides, by binding within a cleft located between the ribosomal protein L11 and helices 43 and 44 of the 23S rRNA, overlap with the position of domain V of EF-G, thus explaining how this class of drugs perturbs translation factor binding to the ribosome. The presence of Micro leads to additional density for the C-terminal domain (CTD) of L7, adjacent to and interacting with L11. The results suggest that L11 acts as a molecular switch to control L7 binding and plays a pivotal role in positioning one L7-CTD monomer on the G' subdomain of EF-G to regulate EF-G turnover during protein synthesis.     

Crosslinking of translation factor EF-G to proteins of the bacterial ribosome before and after translocation

Elongation factor G (EF-G) promotes the translocation of tRNA and mRNA in the central cavity of the ribosome following the addition of each amino acid residue to a growing polypeptide chain. tRNA/mRNA translocation is coupled to GTP hydrolysis, catalyzed by EF-G and activated by the ribosome. In this study we probed EF-G interactions with ribosomal proteins (r-proteins) of the bacterial ribosome, by using a combination of chemical crosslinking, immunoblotting and mass spectroscopy analyses. We identified three bacterial r-proteins (L7/L12, S12 and L6) crosslinked to specific residues of EF-G in three of its domains (G', 3 and 5, respectively). EF-G crosslinks to L7/L12 and S12 were indistinguishable when EF-G was trapped on the ribosome before or after tRNA/mRNA translocation had occurred, whereas a crosslink between EF-G and L6 formed with greater efficiency before translocation had occurred. EF-G crosslinked to L7/L12 was capable of catalyzing multiple rounds of GTP hydrolysis, whereas EF-G crosslinked to S12 was inactive in GTP hydrolysis. These results imply that during the GTP hydrolytic cycle EF-G must detach from S12 within the central cavity of the ribosome, while EF-G can remain associated with L7/L12 located on one of the peripheral stalks of the ribosome. This mechanism may ensure that a single GTP molecule is hydrolyzed for each tRNA/mRNA translocation event.     

Stimulation of the GTPase activity of translation elongation factor G by ribosomal protein L7/12

Elongation factors (EFs) Tu and G are GTPases that have important functions in protein synthesis. The low intrinsic GTPase activity of both factors is strongly stimulated on the ribosome by unknown mechanisms. Here we report that isolated ribosomal protein L7/12 strongly stimulates GTP hydrolysis by EF-G, but not by EF-Tu, indicating a major contribution of L7/12 to GTPase activation of EF-G on the ribosome. The effect is due to the acceleration of the catalytic step because the rate of GDP-GTP exchange on EF-G, as measured by rapid kinetics, is much faster than the steady-state GTPase rate. The unique, highly conserved arginine residue in the C-terminal domain of L7/12 is not essential for the activation, excluding an "arginine finger"-type mechanism. L7/12 appears to function by stabilizing the GTPase transition state of EF-G.     

Control of phosphate release from elongation factor G by ribosomal protein L7/12

Ribosomal protein L7/12 is crucial for the function of elongation factor G (EF-G) on the ribosome. Here, we report the localization of a site in the C-terminal domain (CTD) of L7/12 that is critical for the interaction with EF-G. Single conserved surface amino acids were replaced in the CTD of L7/12. Whereas mutations in helices 5 and 6 had no effect, replacements of V66, I69, K70, and R73 in helix 4 increased the Michaelis constant (KM) of EF-G.GTP for the ribosome, suggesting an involvement of these residues in EF-G binding. The mutations did not appreciably affect rapid single-round GTP hydrolysis and had no effect on tRNA translocation on the ribosome. In contrast, the release of inorganic phosphate (Pi) from ribosome-bound EF-G.GDP.Pi was strongly inhibited and became rate-limiting for the turnover of EF-G. The control of Pi release by interactions between EF-G and L7/12 appears to be important for maintaining the conformational coupling between EF-G and the ribosome for translocation and for timing the dissociation of the factor from the ribosome.     

Preparation and characterization of two monoclonal antibodies against different epitopes in Escherichia coli ribosomal protein L7/L12

Two monoclonal antibodies with specificities for Escherichia coli 50 S ribosomal subunit protein L7/L12 were isolated. The antibodies and Fab fragments thereof were purified by affinity chromatography using solid-phase coupled L7/L12 protein as the immunoadsorbent. The two antibodies were shown to recognize different epitopes; one in the N-terminal and the other in the C-terminal domain of protein L7/L12. Both intact antibodies strongly inhibited polyuridylic acid-directed polyphenylalanine synthesis, ribosome-dependent GTPase activity, and the binding of elongation factor EF-G to the ribosome. Ratios of antibody to ribosome of 4:1 or less were effective in inhibiting these activities. Neither antibody prevented the association of ribosomal subunits to form 70 S ribosomes. The Fab fragments showed similar effects.     

The ribosomal domain of the bacterial release factors. The carboxyl-terminal domain of the dimer of Escherichia coli ribosomal protein L7/L12 located in the body of the ribosome is important for release factor interaction.

1. Polyclonal antibodies (pAb 1-73 and pAb 26-120) have been raised against both an N-terminal fragment of Escherichia coli ribosomal protein L7/L12 (amino acids 1-73), and a fragment lacking part of the N-terminal domain (amino acids 26-120). 2. Only pAb 26-120 inhibited release-factor-dependent in vitro termination functions on the ribosome. This antibody binds over the length of the stalk of the large subunit of the ribosome as determined by immune electron microscopy, thereby not distinguishing between the C-terminal domains of the two L7/L12 dimers, those in the stalk or those in the body of the subunit. 3. A monoclonal antibody against an epitope of the C-terminal two thirds of the protein (mAb 74-120), which binds both to the distal tip of the stalk as well as to a region at its base, reflecting the positions of the two dimers is strongly inhibitory of release factor function. 4. A monoclonal antibody against an epitope of the N-terminal fragment of L7/L12 (mAb 1-73), previously shown to remove the dimer of L7/L12 in the 50S subunit stalk but still bind to the body of the particle, partially inhibited release-factor-mediated events. 5. The mAb 74-120 inhibited in vitro termination with a similar profile when the stalk dimer of L7/L12 was removed with mAb 1-73, indicating that the body L7/L12 dimer, and in particular its C-terminal domains, are important for release factor/ribosome interaction. 6. The two release factors have subtle differences in their binding domains with respect to L7/L12.     

Single-molecule structural dynamics of EF-G--ribosome interaction during translocation

EF-G catalyzes translocation of mRNA and tRNAs within the ribosome during protein synthesis. Detection of structural states in the reaction sequence that are not highly populated can be facilitated by studying the process one molecule at a time. Here we present single-molecule studies of translocation showing that, for ribosomes engaged in poly(Phe) synthesis, fluorescence resonance energy transfer (FRET) between the G' domain of EF-G and the N-terminal domain of ribosomal protein L11 occurs within two rapidly interconverting states, having FRET efficiencies of 0.3 and 0.6. The antibiotic fusidic acid increases the population of the 0.6 state, indicating that it traps the ribosome.EF-G complex in a preexisting conformation formed during translation. Only the 0.3 state is observed when poly(Phe) synthesis is prevented by omission of EF-Tu, or in studies on vacant ribosomes. These results suggest that the 0.6 state results from the conformational lability of unlocked ribosomes formed during translocation. An idling state, possibly pertinent to regulation of protein synthesis, is detected in some ribosomes in the poly(Phe) system.     

The function of conserved amino acid residues adjacent to the effector domain in elongation factor G

Bacterial elongation factor G (EF-G) physically associates with translocation-competent ribosomes and facilitates transition to the subsequent codon through the coordinate binding and hydrolysis of GTP. In order to investigate the amino acid positions necessary for EF-G functions, a series of mutations were constructed in the EF-G structural gene (fusA) of Escherichia coli, specifically at positions flanking the effector domain. A mutated allele was isolated in which the wild-type sequence from codons 29 to 47 ("EFG2947") was replaced with a sequence encoding 28 amino acids from ribosomal protein S7. This mutated gene was unable to complement a fusAts strain when supplied in trans at the nonpermissive temperature. In vitro biochemical analysis demonstrated that nucleotide crosslinking was unaffected in EFG2947, while ribosome binding appeared to be completely abolished. A series of point mutations created within this region, encoding L30A, Y32A, H37A, and K38A were shown to give rise to fully functional proteins, suggesting that side chains of these individual residues are not essential for EF-G function. A sixth mutant, E41A, was found to inefficiently rescue growth in a fusAts background, and was also unable to bind ribosomes normally in vitro. In contrast E41Q could restore growth at the nonpermissive temperature. These results can be explained within the context of a three-dimensional model for the effector region of EF-G. This model indicates that the effector domain contains a negative potential field that may be important for ribosome binding.     

A method of focused classification, based on the bootstrap 3D variance analysis, and its application to EF-G-dependent translocation

The bootstrap-based method for calculation of the 3D variance in cryo-EM maps reconstructed from sets of their projections was applied to a dataset of functional ribosomal complexes containing the Escherichia coli 70S ribosome, tRNAs, and elongation factor G (EF-G). The variance map revealed regions of high variability in the intersubunit space of the ribosome: in the locations of tRNAs, in the putative location of EF-G, and in the vicinity of the L1 protein. This result indicated heterogeneity of the dataset. A method of focused classification was put forward in order to sort out the projection data into approximately homogenous subsets. The method is based on the identification and localization of a region of high variance that a subsequent classification step can be focused on by the use of a 3D spherical mask. After initial classification, template volumes are created and are subsequently refined using a multireference 3D projection alignment procedure. In the application to the ribosome dataset, the two resulting structures were interpreted as resulting from ribosomal complexes with bound EF-G and an empty A site, or, alternatively, from complexes that had no EF-G bound but had both A and P sites occupied by tRNA. The proposed method of focused classification proved to be a successful tool in the analysis of the heterogeneous cryo-EM dataset. The associated calculation of the correlations within the density map confirmed the conformational variability of the complex, which could be interpreted in terms of the ribosomal elongation cycle.     

Functional compatibility of elongation factors between mammalian mitochondrial and bacterial ribosomes: characterization of GTPase activity and translation elongation by hybrid ribosomes bearing heterologous L7/12 proteins

The mammalian mitochondrial (mt) ribosome (mitoribosome) is a bacterial-type ribosome but has a highly protein-rich composition. Almost half of the rRNA contained in the bacterial ribosome is replaced with proteins in the mitoribosome. Escherichia coli elongation factor G (EF-G Ec) has no translocase activity on the mitoribosome but EF-G mt is functional on the E.coli ribosome. To investigate the functional equivalency of the mt and E.coli ribosomes, we prepared hybrid mt and E.coli ribosomes. The hybrid mitoribosome containing E.coli L7/12 (L7/12 Ec) instead of L7/12 mt clearly activated the GTPase of EF-G Ec and efficiently promoted its translocase activity in an in vitro translation system. Thus, the mitoribosome is functionally equivalent to the E.coli ribosome despite their distinct compositions. The mt EF-Tu-dependent translation activity of the E.coli ribosome was also clearly enhanced by replacing the C-terminal domain (CTD) of L7/12 Ec with the mt counterpart (the hybrid E.coli ribosome). This strongly indicates that the CTD of L7/12 is responsible for EF-Tu function. These results demonstrate that functional compatibility between elongation factors and the L7/12 protein in the ribosome governs its translational specificity.     

Mechanism and rates of exchange of L7/L12 between ribosomes and the effects of binding EF-G

The ribosomal stalk complex binds and recruits translation factors to the ribosome during protein biosynthesis. In Escherichia coli the stalk is composed of protein L10 and four copies of L7/L12. Despite the crucial role of the stalk, mechanistic details of L7/L12 subunit exchange are not established. By incubating isotopically labeled intact ribosomes with their unlabeled counterparts we monitored the exchange of the labile stalk proteins by recording mass spectra as a function of time. On the basis of kinetic analysis, we proposed a mechanism whereby exchange proceeds via L7/L12 monomers and dimers. We also compared exchange of L7/L12 from free ribosomes with exchange from ribosomes in complex with elongation factor G (EF-G), trapped in the posttranslocational state by fusidic acid. Results showed that binding of EF-G reduces the L7/L12 exchange reaction of monomers by ~27% and of dimers by ~47% compared with exchange from free ribosomes. This is consistent with a model in which binding of EF-G does not modify interactions between the L7/L12 monomers but rather one of the four monomers, and as a result one of the two dimers, become anchored to the ribosome-EF-G complex preventing their free exchange. Overall therefore our results not only provide mechanistic insight into the exchange of L7/L12 monomers and dimers and the effects of EF-G binding but also have implications for modulating stability in response to environmental and functional stimuli within the cell.     

Deletion of C-terminal residues of Escherichia coli ribosomal protein L10 causes the loss of binding of one L7/L12 dimer: ribosomes with one L7/L12 dimer are active

Escherichia coli ribosomal protein L10 binds the two L7/L12 dimers and thereby anchors them to the large ribosomal subunit. C-Terminal deletion variants (Delta10, Delta20, and Delta33 amino acids) of ribosomal protein L10 were constructed in order to define the binding sites for the two L7/L12 dimers and then to make and test ribosomal particles that contain only one of the two dimers. None of the deletions interfered with binding of L10 variants to ribosomal core particles. Deletion of 20 or 33 amino acids led to the inability of the proteins to bind both dimers of protein L7/L12. The L10 variant with deletion of 10 amino acids bound one L7/L12 dimer in solution and when reconstituted into ribosomes promoted the binding of only one L7/L12 dimer to the ribosome. The ribosomes that contained a single L7/L12 dimer were homogeneous by gel electrophoresis where they had a mobility between wild-type 50S subunits and cores completely lacking L7/L12. The single-dimer ribosomal particles supported elongation factor G dependent GTP hydrolysis and protein synthesis in vitro with the same activity as that of two-dimer particles. The results suggest that amino acids 145-154 in protein L10 determine the binding site ("internal-site") for one L7/L12 dimer (the one reported here), and residues 155-164 ("C-terminal-site") are involved in the interaction with the second L7/L12 dimer. Homogeneous ribosomal particles containing a single L7/L12 dimer in each of the distinct sites present an ideal system for studying the location, conformation, dynamics, and function of each of the dimers individually.     

Interaction of thiostrepton and elongation factor-G with the ribosomal protein L11-binding domain

Ribosomal protein L11 and the L11 binding region of ribosomal RNA constitute an important domain involved in active functions of the ribosome during translation. We studied the effects of L11 knock-out and truncation mutations on the structure of the rRNA in this region and on its interactions with a translation elongation factor and the antibiotic thiostrepton. The results indicated that the structure of the L11-binding rRNA becomes conformationally flexible when ribosomes lack the entire L11 protein, but not when the C-terminal domain is present on ribosomes. Probing wild type and mutant ribosomes in the presence of the antibiotic thiostrepton and elongation factor-G (EF-G) rigorously localized the binding cleft of thiostrepton and suggested a role for the rRNA in the L11-binding domain in modulating factor binding. Our results also provide evidence that the structure of the rRNA stabilized by the C-terminal domain of L11 is necessary to stabilize EF-G binding in the post-translocation state, and thiostrepton may modulate this structure in a manner that interferes with the ribosome-EF-G interaction. The implications for recent models of thiostrepton activity and factor interactions are discussed.     

The hinge region of Escherichia coli ribosomal protein L7/L12 is required for factor binding and GTP hydrolysis

A variant form of Escherichia coli ribosomal protein L7/L12 that lacked residues 42 to 52 (L7/L12 Δ42–52) in the hinge region was shown previously to be completely inactive in supporting polyphenylalanine synthesis although it bound to L7/L12 deficient core particles with the normal stoichiometry of four copies per particle (Oleinikov AV, Perroud B, Wang B, Traut RR (1993) J Biol Chem, 268, 917–922). The result suggested that the hinge confers flexibility that is required for activity because the resulting bent conformation allows the distal C-terminal domain to occupy a location on the body of the large ribosomal subunit proximal to the base of the L7/L12 stalk where elongation factors bind. Factor binding to the hinge-truncated variant was tested. As an alternative strategy to deleting residues from the hinge, seven amino acid residues within the putative hinge region were replaced by seven consecutive proline residues in an attempt to confer increased rigidity that might reduce or eliminate the bending of the molecule inferred to be functionally important. This variant, L7/L12: (Pro)₇, remained fully active in protein synthesis. Whereas the binding of both factors in ribosomes containing L7/L12:Δ42–52 was decreased by about 50%, there was no loss of factor binding in ribosomes containing L7/L12:(Pro)₇, as predicted from the retention of protein synthesis activity. The factor:ribosome complexes that contained L7/L12:Δ42–52 had the same low level of GTP hydrolysis as the core particles completely lacking L7/L12 and EF-G did not support translocation measured by the reaction of phe-tRNA bounds in hr Asite with puromycin. It is concluded that the hinge region is required for the functionally productive binding of elongation factors, and the defect in protein synthesis reported previously is due to this defect. The variant produced by the introduction of the putative rigid Pro₇ sequence retains sufficient flexibility for full activity.     

Translation elongation by a hybrid ribosome in which proteins at the GTPase center of the Escherichia coli ribosome are replaced with rat counterparts.

Ribosomal L10-L7/L12 protein complex and L11 bind to a highly conserved RNA region around position 1070 in domain II of 23 S rRNA and constitute a part of the GTPase-associated center in Escherichia coli ribosomes. We replaced these ribosomal proteins in vitro with the rat counterparts P0-P1/P2 complex and RL12, and tested them for ribosomal activities. The core 50 S subunit lacking the proteins on the 1070 RNA domain was prepared under gentle conditions from a mutant deficient in ribosomal protein L11. The rat proteins bound to the core 50 S subunit through their interactions with the 1070 RNA domain. The resultant hybrid ribosome was insensitive to thiostrepton and showed poly(U)-programmed polyphenylalanine synthesis dependent on the actions of both eukaryotic elongation factors 1alpha (eEF-1alpha) and 2 (eEF-2) but not of the prokaryotic equivalent factors EF-Tu and EF-G. The results from replacement of either the L10-L7/L12 complex or L11 with rat protein showed that the P0-P1/P2 complex, and not RL12, was responsible for the specificity of the eukaryotic ribosomes to eukaryotic elongation factors and for the accompanying GTPase activity. The presence of either E. coli L11 or rat RL12 considerably stimulated the polyphenylalanine synthesis by the hybrid ribosome, suggesting that L11/RL12 proteins play an important role in post-GTPase events of translation elongation.     

Inhibition of EF-G dependent GTPase by an aminoterminal fragment of L7/L12.

The E. coli ribosomal proteins L12 and its N-acetylated form L7 were cleaved into an N-terminal and C-terminal fragment of roughly comparable size. The selective cleavage at the lone arginine residue was accomplished by trypsin treatment of the citraconylated proteins, followed by removal of the citraconyl moieties. These fragments, both separately and in combination, were incapable of reconstituting elongation factor G (EF-G) dependent GTPase of CsCl ribosomal cores supplemented with L10. However, incubation of cores containing L10 with the N-terminal fragment prevented the reconstitution of GTPase activity by intact L7/L12. No inhibition was observed when CsCl cores lacking L10 were incubated with the N-terminal fragment followed by addition of a preincubated mixture of L7/L12 and L10. The results indicate that the N-terminal part of L7/L12 is responsible for its ability to bind to 50S ribosomes and that L7/L12 together with L10 form a protein cluster on the ribosome.     

Localization and dynamic behavior of ribosomal protein L30e

The ribosomal protein L30e is an indispensable component of the eukaryotic 80S ribosome, where it is part of the large (60S) ribosomal subunit. Here, we determined the localization of L30e in the cryo-EM map of the 80S wheat germ (wg) ribosome at a resolution of 9.5 A. L30e is part of the interface between large and small subunits, where it dynamically participates in the formation of the two intersubunit bridges eB9 and B4.