Structure and binding mode of a ribosome recycling factor (RRF) from mesophilic bacterium

X-ray and NMR analyses on ribosome recycling factors (RRFs) from thermophilic bacteria showed that they display a tRNA-like L-shaped conformation consisting of two domains. Since then, it has been accepted that domain I, consisting of a three-helix bundle, corresponds to the anticodon arm of tRNA and domain II and a beta/alpha/beta sandwich structure, corresponds to the acceptor arm. In this study, we obtained a RRF from a mesophilic bacterium, Vibrio parahaemolyticus, by gene cloning and carried out an x-ray analysis on it at 2.2 A resolution. This RRF was shown to be active in an in vitro assay system using Escherichia coli polysomes and elongation factor G (EF-G). In contrast, the above-mentioned RRFs from thermophilic bacteria were inactive in such a system. Analysis of the relative orientations between the two domains in the structures of various RRFs, including this RRF from mesophilic bacterium, revealed that domain II rotates about the long axis of the helix bundle of domain I. To elucidate the ribosome binding site of RRF, the peptide fragment (RRF-DI) corresponding to domain I of RRF was expressed and characterized. RRF-DI is bound to 70 S ribosome and the 50 S subunit with an affinity similar to that of wild-type RRF. But it does not bind to the 30 S subunit. These findings caused us to reinvestigate the concept of the mimicry of RRF to tRNA and to propose a new model where domain I corresponds to the acceptor arm of tRNA and domain II corresponds to the anticodon arm. This is just the reverse of a model that is now widely accepted. However, the new model is in better agreement with published biological findings.     

Solution structure of the ribosome recycling factor from Aquifex aeolicus

The solution structure of ribosome recycling factor (RRF) from hyperthermophilic bacterium, Aquifex aeolicus, was determined by heteronuclear multidimensional NMR spectroscopy. Fifteen structures were calculated using restraints derived from NOE, J-coupling, and T1/T2 anisotropies. The resulting structure has an overall L-shaped conformation with two domains and is similar to that of a tRNA molecule. The domain I (corresponding to the anticodon stem of tRNA) is a rigid three alpha-helix bundle. Being slightly different from usual coiled-coil arrangements, each helix of domain I is not twisted but straight and parallel to the main axis. The domain II (corresponding to the portion with the CCA end of tRNA) is an alpha/beta domain with an alpha-helix and two beta-sheets, that has some flexible regions. The backbone atomic root-mean-square deviation (rmsd) values of both domains were 0.7 A when calculated separately, which is smaller than that of the molecule as a whole (1.4 A). Measurement of 15N-[1H] NOE values show that the residues in the corner of the L-shaped molecule are undergoing fast internal motion. These results indicate that the joint region between two domains contributes to the fluctuation in the orientation of two domains. Thus, it was shown that RRF remains the tRNA mimicry in solution where it functions.     

Crystal structure of the ribosome recycling factor from Escherichia coli

We have determined the crystal structure of the Escherichia coli ribosome recycling factor (RRF), which catalyzes the disassembly of the termination complex in protein synthesis. The L-shaped molecule consists of two domains: a triple-stranded antiparallel coiled-coil and an alpha/beta domain. The coil domain has a cylindrical shape and negatively charged surface, which are reminiscent of the anticodon arm of tRNA and domain IV of elongation factor EF-G. We suggest that RRF binds to the ribosomal A-site through its coil domain, which is a tRNA mimic. The relative position of the two domains is changed about an axis along the hydrophobic cleft in the hinge where the alkyl chain of a detergent molecule is bound. The tRNA mimicry and the domain movement observed in RRF provide a structural basis for understanding the role of RRF in protein synthesis.     

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.     

Specific interaction between EF-G and RRF and its implication for GTP-dependent ribosome splitting into subunits

After termination of protein synthesis, the bacterial ribosome is split into its 30S and 50S subunits by the action of ribosome recycling factor (RRF) and elongation factor G (EF-G) in a guanosine 5′-triphosphate (GTP)-hydrolysis-dependent manner. Based on a previous cryo-electron microscopy study of ribosomal complexes, we have proposed that the binding of EF-G to an RRF-containing posttermination ribosome triggers an interdomain rotation of RRF, which destabilizes two strong intersubunit bridges (B2a and B3) and, ultimately, separates the two subunits. Here, we present a 9-Å (Fourier shell correlation cutoff of 0.5) cryo-electron microscopy map of a 50S·EF-G·guanosine 5′-[(βγ)-imido]triphosphate·RRF complex and a quasi-atomic model derived from it, showing the interaction between EF-G and RRF on the 50S subunit in the presence of the noncleavable GTP analogue guanosine 5′-[(βγ)-imido]triphosphate. The detailed information in this model and a comparative analysis of EF-G structures in various nucleotide- and ribosome-bound states show how rotation of the RRF head domain may be triggered by various domains of EF-G. For validation of our structural model, all known mutations in EF-G and RRF that relate to ribosome recycling have been taken into account. More importantly, our results indicate a substantial conformational change in the Switch I region of EF-G, suggesting that a conformational signal transduction mechanism, similar to that employed in transfer RNA translocation on the ribosome by EF-G, translates a large-scale movement of EF-G's domain IV, induced by GTP hydrolysis, into the domain rotation of RRF that eventually splits the ribosome into subunits.     

Characteristic domain motion in the ribosome recycling factor revealed by 15N NMR relaxation experiments and molecular dynamics simulations

The backbone dynamics of ribosome recycling factor (RRF) from Escherichia coli in water were characterized by (15)N NMR relaxation analysis and molecular dynamics (MD) simulation. RRF is composed of two domains connected by a joint region that consists of two peptide chains, such that the overall structure seems to mimic that of tRNA. MD trajectories indicated that the relative orientation of domains varies on the nanosecond time scale. We analyzed the observed (15)N T(1), T(2), and NOE using an extended model-free spectral density function in which the domain motions with a nanosecond time scale were considered. At 30 degrees C, the order parameters of slow motion () were determined to be approximately 0.9 for domain I and 0.7 for domain II, respectively. These values indicate that domain I is nearly fixed on the molecular diffusion frame, and domain II is wobbling in a cone for which the semi-angle is about 30 degrees.     

Heterologous expression of Aquifex aeolicus ribosome recycling factor in Escherichia coli is dominant lethal by forming a complex that lacks functional co-ordination for ribosome disassembly

Recycling the post-termination ribosomal complex requires the co-ordinated effort of the ribosome, ribosome recycling factor (RRF) and elongation factor EF-G. Although Aquifex aeolicus RRF (aaRRF) binds Escherichia coli ribosomes as efficiently as E. coli RRF, the resulting complex is non-functional and dominant lethal in E. coli, even in the presence of homologous A. aeolicus EF-G. These findings suggest that the E. coli post-termination ribosomal complex with aaRRF lacks functional co-ordination with EF-G required for ribosome recycling. A chimeric EF-G (E. coli domains I-III, A. aeolicus domains IV-V) or an A. aeolicus EF-G with distinct mutations in the domain I-II interface could activate aaRRF. Furthermore, novel mutations that localize to one surface of the L-shape structure of aaRRF restored activity in E. coli. These aaRRF mutations are spatially distinct from mutations previously described and suggest a novel active centre for coupling EF-G's G domain motor action to ribosome disassembly.     

Crystal structure combined with genetic analysis of the Thermus thermophilus ribosome recycling factor shows that a flexible hinge may act as a functional switch

Ribosome recycling factor (RRF), in concert with elongation factor EF-G, is required for disassembly of the posttermination complex of the ribosome after release of polypeptides. The crystal structure of Thermus thermophilus RRF was determined at 2.6 A resolution. It is a tRNA-like L-shaped molecule consisting of two domains: a long three-helix bundle (domain 1) and a three-layer beta/alpha/beta sandwich (domain 2). Although the individual domain structures are similar to those of Thermotoga maritima RRF (Selmer et al., Science, 1999, 286:2349-2352), the interdomain angle differs by 33 degrees in two molecules, suggesting that the hinge between two domains is potentially flexible and responsive to different conditions of crystal packing. The hinge connects hydrophobic junctions of domains 1 and 2. The structure-based genetic analysis revealed the strong correlation between the hinge flexibility and the in vivo function of RRF. First, altering the hinge flexibility by making alanine or serine substitutions for large-size residues conserved at the hinge loop and nearby in domain 1 frequently gave rise to gain of function except a Pro residue conserved at the hinge loop. Second, the hinge defect resulting from a too relaxed hinge structure can be compensated for by secondary alterations in domain 1 that seem to increase the hydrophobic contact between domain 1 and the hinge loop. These results show that the hinge flexibility is vital for the function of RRF and that the steric interaction between the hinge loop and domains 1 and 2 restricts the interdomain angle and/or the hinge flexibility. These results indicate that RRF possesses an architectural difference from tRNA regardless of a resemblance to tRNA shape: RRF has a "gooseneck" elbow, whereas the tRNA elbow is rigid, and the direction of flex of RRF and tRNA is at a nearly right angle to each other. Moreover, surface electrostatic potentials of the two RRF proteins are dissimilar and do not mimic the surface potential of tRNA or EF-G. These properties will add a new insight into RRF, suggesting that RRF is more than a simple tRNA mimic.     

Recycling of the Posttermination Complexes of Mycobacterium smegmatis and Escherichia coli Ribosomes Using Heterologous Factors

In eubacteria, ribosome recycling factor (RRF) and elongation factor G (EFG) function together to dissociate posttermination ribosomal complexes. Earlier studies, using heterologous factors from Mycobacterium tuberculosis in Escherichia coli revealed that specific interactions between RRF and EFG are crucial for their function in ribosome recycling. Here, we used translation factors from E. coli, Mycobacterium smegmatis and M. tuberculosis, and polysomes from E. coli and M. smegmatis, and employed in vivo and in vitro experiments to further understand the role of EFG in ribosome recycling. We show that E. coli EFG (EcoEFG) recycles E. coli ribosomes with E. coli RRF (EcoRRF), but not with mycobacterial RRFs. Also, EcoEFG fails to recycle M. smegmatis ribosomes with either EcoRRF or mycobacterial RRFs. On the other hand, mycobacterial EFGs recycle both E. coli and M. smegmatis ribosomes with either of the RRFs. These observations suggest that EFG establishes distinct interactions with RRF and the ribosome to carry out ribosome recycling. Furthermore, the EFG chimeras generated by swapping domains between mycobacterial EFGs and EcoEFG suggest that while the residues needed to specify the EFG interaction with RRF are located in domains IV and V, those required to specify its interaction with the ribosome are located throughout the molecule.     

Elongation factor G participates in ribosome disassembly by interacting with ribosome recycling factor at their tRNA-mimicry domains

Elongation factor G (EF-G) is a G protein with motor function that drives two target molecules, a tRNA in the translating ribosome and the ribosome recycling factor (RRF) in the post-termination complex. How G protein motor action is transmitted to RRF is unknown. Thermus thermophilus RRF is nonfunctional in Escherichia coli. It became functional upon introducing a plasmid expressing E. coli EF-G with surface changes in its tRNA-mimic domain or by replacing the E. coli EF-G tRNA-mimic domain by the Thermus domain. Thermus RRF could also be activated by introducing surface substitutions in its anticodon arm-mimic region. These gain-of-function phenotypes depend on the combination of heterologous EF-G and RRF alleles. These mutational studies suggest that EF-G motor action is transmitted to RRF by specific surface contacts between the domains that mimic the anticodon arm.     

Molecular flexibility of Mycobacterium tuberculosis ribosome recycling factor and its functional consequences: An exploration involving mutants

Internal mobility of the two domain molecule of ribosome recycling factor (RRF) is known to be important for its action. Mycobacterium tuberculosis RRF does not complement E. coli for its deficiency of RRF (in the presence of E. coli EF-G alone). Crystal structure had revealed higher rigidity of the M. tuberculosis RRF due to the presence of additional salt bridges between domains. Two inter-domain salt bridges and one between the linker region and the domain containing C-terminal residues were disrupted by appropriate mutations. Except for a C-terminal deletion mutant, all mutants showed RRF activity in E. coli when M. tuberculosis EF-G was also co-expressed. The crystal structures of the point mutants, that of the C-terminal deletion mutant and that of the protein grown in the presence of a detergent, were determined. The increased mobility resulting from the disruption of the salt bridge involving the hinge region allows the appropriate mutant to weakly complement E. coli for its deficiency of RRF even in the absence of simultaneous expression of the mycobacterial EF-G. The loss of activity of the C-terminal deletion mutant appears to be partly due to the rigidification of the molecule consequent to changes in the hinge region.     

Structural Insights into ribosome recycling factor interactions with the 70S ribosome

At the end of translation in bacteria, ribosome recycling factor (RRF) is used together with elongation factor G to recycle the 30S and 50S ribosomal subunits for the next round of translation. In x-ray crystal structures of RRF with the Escherichia coli 70S ribosome, RRF binds to the large ribosomal subunit in the cleft that contains the peptidyl transferase center. Upon binding of either E. coli or Thermus thermophilus RRF to the E. coli ribosome, the tip of ribosomal RNA helix 69 in the large subunit moves away from the small subunit toward RRF by 8 Å, thereby disrupting a key contact between the small and large ribosomal subunits termed bridge B2a. In the ribosome crystals, the ability of RRF to destabilize bridge B2a is influenced by crystal packing forces. Movement of helix 69 involves an ordered-to-disordered transition upon binding of RRF to the ribosome. The disruption of bridge B2a upon RRF binding to the ribosome seen in the present structures reveals one of the key roles that RRF plays in ribosome recycling, the dissociation of 70S ribosomes into subunits. The structures also reveal contacts between domain II of RRF and protein S12 in the 30S subunit that may also play a role in ribosome recycling.     

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.     

Expression in Escherichia coli, purification and characterization of Thermoanaerobacter tengcongensis ribosome recycling factor

A very promising approach to understanding the mechanism of protein thermostability is to investigate the structure-function relationship of homologous proteins with different thermostabilities. Ribosome recycling factor (RRF), which is an essential factor for protein synthesis in bacteria, may be a good candidate for such study. In this report, a ribosome recycling factor from Thermoanaerobacter tengcongensis was expressed and characterized. This protein contains 184 residues, shows 51.4% identity to that of Escherichia coli RRF, and has very strong antigenic cross-reactivity with antibody to E. coli RRF. In vivo activity assay shows that weak residual activity may remain in TteRRF in E. coli cells. Circular dichroism spectral analysis shows that TteRRF has a very similar secondary structure to that of E. coli RRF, implying that they have similar tertiary structures. However, their thermostabilities are significantly different. To find which domain of RRF is mainly responsible for maintaining stability, TteDI/EcoDII and EcoDI/TteDII RRF chimeras were created. Their domain I and domain II are from E. coli and T. tengcongensis RRFs, respectively. The results of GdnHCl and heat induced denaturation of the chimeric RRFs suggest that the domain I plays a major role in maintaining the stability of the RRF molecule.     

Mutations influencing the frr gene coding for ribosome recycling factor (RRF)

A total of 52 null, six reversion, and five silent mutations of frr (the gene encoding for ribosome recycling factor (RRF)) of Escherichia coli are discussed along with 12 temperature-sensitive (ts) mutations and 14 intergenic suppressor strains of ts RRF. The null mutations were classified into six different categories. A computer-based secondary structure analysis showed three domains; domain A which has the N-terminal helix, domain B which contains coil, alpha-helix and beta-strand structure, and domain C which is a C-terminal helix. The ts mutations fell into domains A and C but not in domain B. More than a half of the null mutations fell into domain B while the silent mutations fell outside domain B. Substitution of Arg132 in domain C by other amino acids was observed among five independently isolated null mutants. It is suggested that domain B is important for maintaining the RRF structure, while the region including Arg132 is one of the active sites. A total of 14 intergenic suppressor strains of ts RRF were grouped into four categories, depending on which temperature-sensitive alleles were suppressed.     

Mechanism for the disassembly of the posttermination complex inferred from cryo-EM studies

Ribosome recycling, the disassembly of the posttermination complex after each round of protein synthesis, is an essential step in mRNA translation, but its mechanism has remained obscure. In eubacteria, recycling is catalyzed by RRF (ribosome recycling factor) and EF-G (elongation factor G). By using cryo-electron microscopy, we have obtained two density maps, one of the RRF bound posttermination complex and one of the 50S subunit bound with both EF-G and RRF. Comparing the two maps, we found domain I of RRF to be in the same orientation, while domain II in the EF-G-containing 50S subunit is extensively rotated (approximately 60 degrees) compared to its orientation in the 70S complex. Mapping the 50S conformation of RRF onto the 70S posttermination complex suggests that it can disrupt the intersubunit bridges B2a and B3, and thus effect a separation of the two subunits. These observations provide the structural basis for the mechanism by which the posttermination complex is split into subunits by the joint action of RRF and EF-G.     

Ribosome recycling revisited

Ribosome recycling involves the coordinated action of the ribosome recycling factor (RRF), elongation factor EF-G and initiation factor IF3 to disassemble the post-termination complex, recycling the components for the next round of translation. The crystal structure of domain I of RRF (RRF-DI) in complex with the large ribosomal subunit from the eubacteria Deinococcus radiodurans at high resolution reveals the nature and details of the interactions between this protein factor and rRNA/protein components of the ribosome. Universally conserved arginine residues within the RRF-DI establish important interactions with nuleotides of the 23S rRNA, explaining why mutations at these positions abolish factor binding. Furthermore, in conjunction with cryo-EM reconstruction, the X-ray analysis provides a structural complement to the recent biochemical data, offering additional insight into the mechanism of ribosome recycling.     

Ribosome recycling revisited

Ribosome recycling involves the coordinated action of the ribosome recycling factor (RRF), elongation factor EF-G, and the initiation factor IF3 to disassemble the posttermination complex, recycling the components for the next round of translation. The crystal structure of domain I of RRF (RRF-DI) in complex with the large ribosomal subunit from the eubacteria Deinococcus radiodurans at a high resolution reveals the nature and details of the interactions between this protein factor and the rRNA/protein components of the ribosome. Universally conserved arginine residues within the RRF-DI establish important interactions with nucleotides of the 23S rRNA, thus explaining why mutations at these positions abolish factor binding. Furthermore, in conjunction with cryo-EM reconstruction, the X-ray analysis provides a structural complement to the recent biochemical data, offering additional insight into the mechanism of ribosome recycling. Published in Russian in Molekulyarnaya Biologiya, 2006, Vol. 40, No. 4, pp. 742–750. The text was submitted by the authors in English.     

Structure and Dynamics of Ribosomal Protein L12: An Ensemble Model Based on SAXS and NMR Relaxation

Ribosomal protein L12 is a two-domain protein that forms dimers mediated by its N-terminal domains. A 20-residue linker separates the N- and C-terminal domains. This linker results in a three-lobe topology with significant flexibility, known to be critical for efficient translation. Here we present an ensemble model of spatial distributions and correlation times for the domain reorientations of L12 that reconciles experimental data from small-angle x-ray scattering and nuclear magnetic resonance. We generated an ensemble of L12 conformations in which the structure of each domain is fixed but the domain orientations are variable. The ensemble reproduces the small-angle x-ray scattering data and the optimized correlation times of its reorientational eigenmodes fit the ¹⁵N relaxation data. The ensemble model reveals intrinsic conformational properties of L12 that help explain its function on the ribosome. The two C-terminal domains sample a large volume and extend further away from the ribosome anchor than expected for a random-chain linker, indicating that the flexible linker has residual order. Furthermore, the distances between each C-terminal domain and the anchor are anticorrelated, indicating that one of them is more retracted on average. We speculate that these properties promote the function of L12 to recruit translation factors and control their activity on the ribosome.