Movatterモバイル変換

[0]ホーム
URL:

Skip to main content
NCBI home page
Search in PMCSearch
As a library, NLM provides access to scientific literature. Inclusion in an NLM database does not imply endorsement of, or agreement with, the contents by NLM or the National Institutes of Health.
Learn more:PMC Disclaimer | PMC Copyright Notice
NIHPA Author Manuscripts logo
. Author manuscript; available in PMC: 2017 Dec 6.

Key intermediates in ribosome recycling visualized by time-resolved cryo-electron microscopy

Ziao Fu1,6,Sandip Kaledhonkar2,6,Anneli Borg3,6,Ming Sun4,Bo Chen4,Robert A Grassucci2,5,Mans Ehrenberg3,Joachim Frank2,4,5,7,*
1Integrated Program in Cellular, Molecular, and Biomedical Studies, Columbia University College of Physicians and Surgeons, New York, NY 10032, USA
2Department of Biochemistry and Molecular Biophysics, Columbia University New York, NY 10027, USA
3Department of Cell and Molecular Biology, Uppsala University, Sweden
4Department of Biological Sciences, Columbia University, New York, NY 10027, USA
5Howard Hughes Medical Institute, Columbia University, New York, NY 10032, USA
*

Correspondence:jf2192@cumc.columbia.edu

6

Co-first author

7

Lead Contact

Issue date 2016 Dec 6.

PMCID: PMC5143168  NIHMSID: NIHMS823584  PMID:27818103
The publisher's version of this article is available atStructure

Abstract

Upon encountering a stop codon on messenger RNA (mRNA), polypeptide synthesis on the ribosome is terminated by release factors, and the ribosome complex, still bound with mRNA and P site-bound tRNA (post-termination complex, PostTC), is split into ribosomal subunits, ready for a new round of translational initiation. Separation of post-termination ribosomes into subunits, or “ribosome recycling”, is promoted by the joint action of ribosome recycling factor (RRF) and elongation factor G (EF-G) in a GTP hydrolysis-dependent manner. Here we used a mixing-spraying based method of time-resolved cryo-electron microscopy (cryo-EM) to visualize the short-lived intermediates of the recycling process. The two complexes that contain (1) both RRF and EF-G bound to the PostTC or (2) deacylated tRNA bound to the 30S subunit are of particular interest. Our observations of the native form of these complexes demonstrate the strong potential of time-resolved cryo-EM for visualizing previously unobservable transient structures.

eTOC Blurb

In this study Fu, Kaledhonkar, Borg et al. visualize the short-lived post-termination 70SR•RF•EF-G complex before the onset of recycling, and show that IF3 displaces tRNA on the 30S subunit in one possible recycling pathway. This study thus demonstrates the potential of time-resolved cryo-EM in depicting short-lived states of molecular interactions.

Introduction

Ribosome recycling is one of four major stages of protein synthesis, following initiation, elongation and termination(Ramakrishnan, 2002). After termination, an mRNA-containing ribosome complex with a deacylated tRNA in the P site is targeted for recycling by the concerted action of ribosome recycling factor (RRF) and elongation factor G (EF-G). During recycling the post-termination ribosome is split into its large (50S) and small (30S) subunit in a GTP hydrolysis-dependent manner for a new round of initiation (Hirashima and Kaji, 1973;Zavialov et al., 2005). Regarding the molecular mechanism of ribosome recycling, it was found that the shape of the crystal structure of RRF fromThermotoga maritima superimposes almost perfectly with that of a tRNA (Selmer et al., 1999). This finding prompted speculations that RRF binds to the A site of the ribosome as a peptidyl-tRNA mimic and is then translocated by EF-G by a mechanism akin to canonical mRNA-tRNA translocation. However, site-directed hydroxyl radical cleavage assays (Lancaster et al., 2002), cryo-electron microscopy (cryo-EM) (Agrawal et al., 2004;Barat et al., 2007;Gao et al., 2005;Yokoyama et al., 2012) and X-ray crystallography (Borovinskaya et al., 2007;Dunkle et al., 2011;Wilson et al., 2004) of ribosome-RRF complexes indicated that the position of RRF bound to the ribosome is distinct from that of an A-site tRNA. Binding of RRF stabilizes a fully rotated state of the ribosome, with the P-site tRNA moved to the hybrid P/E binding state (Dunkle et al., 2011).

Domain I of RRF is important for its binding to the ribosome. Domain II of RRF is connected to domain I by two flexible linkers, which permit relative domain rotation (Weixlbaumer et al., 2007). Comparison of the various crystal structures of RRF (Kim et al., 2000;Nakano et al., 2003;Toyoda et al., 2000) with its solution structure (Yoshida et al., 2001), NMR relaxation experiments (Yoshida et al., 2003) and a study of mutations around the hinge region (Toyoda et al., 2000) have demonstrated that such a rotation does take place and suggested that the flexibility of the hinge region is functionally important. In a cryo-EM study it has been shown that whenThermus thermophilus RRF (ttRRF) binds together withEscherichia coli (E. coli) EF-G to theE. coli ribosome (Yokoyama et al., 2012), domain II of ttRRF is rotated by a large angle. The physiological relevance of these findings is uncertain as this heterologous combination has authentic recycling activity neitherin vitro (Raj et al., 2005) norin vivo (Fujiwara et al., 1999;Ito et al., 2002;Toyoda et al., 2000). Another player in ribosome recycling is initiation factor 3 (IF3). After separation of the ribosomal subunits this factor binds to the 30S subunit to prevent its rebinding to the 50S subunit. According to one model of ribosome recycling (Hirokawa et al., 2006), in which the tRNA already leaves the PostTC upon action by RRF and EF-G, this is the only role of IF3. In another model, the deacylated tRNA stays bound to the 30S subunit until the ribosome has been split, and IF3 has the additional role to accelerate dissociation of deacylated tRNA, which in turn speeds up dissociation of mRNA from the 30S subunit (Karimi et al., 1999).

The mechanism of action of RRF and EF-G in recycling of the bacterial ribosome poses a major puzzle which has defied exploration by existing techniques of structure research. Ribosome recycling entails transitions between structural intermediates of the ribosome, driven by domain movements of EF-G and RRF, which, in turn, are driven by the free energy liberated upon EF-G-catalyzed GTP hydrolysis (Savelsbergh et al., 2009). This sequence of events implies the existence of short-lived ribosome structures that unlock the interactions of the ribosomal subunits and allow their rapid separation by the concerted action of EF-G and RRF. It would, in principle, be possible to visualize one of these structures by locking the recycling process in an intermediate state, e.g., by replacing GTP with one of its non-hydrolyzable analogues. The caveat here is that the relevance of the visualized structure would be difficult to assess in this process. Furthermore, all such efforts to artificially lock the ribosome in an intermediate recycling state with active EF-G and RRF of the same species have failed. The only 70S ribosome visualized in complex with both of these translation factors is the above mentioned complex with EF-G from E. coli and RRF fromT. thermophiles (Yokoyama et al., 2012).

This short outline may serve to underline the urgent need for an experimental method that allows both the determination of structures that appear in sequential transients and the examination of the kinetic order in which they appear. If successful, this method would be applicable not only to the study of ribosome recycling but also of a variety of other fast processes presently out of reach for structural and dynamics studies such as initiation of mRNA translation, peptide elongation, termination of translation and recycling of class-1 release factor by a class-2 release factor.

Recent kinetic experiments (Borg et al., 2015) have shown that ribosome recycling occurs in the sub-second time range, offering hope that reaction intermediates can be captured by a recently established technique of time-resolved cryo-EM, which operates in the same time frame (Chen et al.;Lu et al., 2009;Shaikh et al., 2014). In this mixing-spraying setup of time-resolved cryo-EM, a monolithic silicon chip is used to mix and react two solutions for pre-set times, followed by spraying the reaction product onto an EM grid that is being plunged into liquid cryogen. In the present study we have used this method to study the ribosome recycling driven by native RRF, EF-G and GTP. Our results, obtained by single-particle reconstruction aided by 3D maximum likelihood classification, unequivocally show RRF and EF-G bound together to an authentic post-termination complex within the time window predicted from the kinetic analysis.

Results

Biochemical characterization and computer simulations

In a recent study we performed real-time ribosome splitting experiments by the stopped-flow technique with Rayleigh light scattering detection at 37°C (Borg et al., 2015). By complementing these experiments with measurements of EF-G-dependent GTP hydrolysis, the complete kinetic mechanism of ribosome recycling was identified and its rate constants at 37 °C were estimated (Table S1 andFig. S1). In these experiments it was found that EF-G competes with RRF for binding to the post-termination ribosome, leading to inhibition of recycling and futile GTP hydrolysis cycles at comparatively high EF-G concentration. Ribosome splitting is efficient and takes place in the milliseconds range. According to the ribosome splitting model proposed by Borg et al. (Borg et al., 2015) RRF binding to the 70S post-termination ribosome is followed by EF-G binding to the PoTC•RRF complex, which in turn triggers the splitting of the subunits (see the simplified reaction scheme inFigure 1A). As the time-resolved cryo-EM apparatus is operated at room temperature, we repeated the previous kinetic experiments (Borg et al., 2015) also at 20 °C (Table S2 andFig. S2). Most association and dissociation rate constants were between three- and tenfold smaller at 20 °C than at 37 °C, and the maximal splitting rate (kmax) was estimated as 1.3 s−1 at 20 °C and 25 s−1 at 37 °C. As it turned out, the actual cryo-EM experiments were performed at ~26 °C, and we used the Arrhenius equation (see Eqs. 1 and 2) to estimate the rate constants at this temperature from the 20 °C and 37 °C data (Tables S1 and S2). These rate constants show that the highest value of the maximal PoTC•RRF•EF-G fraction can be obtained by pre-incubating the post-termination complex with high concentration of RRF. In this way, idling of EF-G on the factor-free post-termination complex can be curbed by reducing the frequency with which ribosomes enter the vertical branch of the reaction scheme (Figure 1A). At 45 μM [RRF] and 10 μM [EF-G] the fraction of the PoTC•RRF•EF-G complex peaked at around 100 ms, constituting almost 70% of the ribosomes (seeFigure 1). We therefore used the closest available reaction chip, with 140 ms total reaction time, for the time-resolved cryo-EM experiment. To confirm the completeness of the recycling reaction, we performed a long-incubation experiment (see Experimental Procedures).

Figure 1. The recycling process.

Figure 1

(A) Simplified scheme for EF-G- and RRF-dependent recycling of the post-termination complex. The 26°C rate constant estimates were interpolated from experimental estimates of the corresponding rate constants at 20 °C and 37 °C using the Arrhenius equation (Table S2). [RRF], concentration of RRF; [G], concentration of EF-G; kRRF = 6.6 μM−1s−1, qRRF = 14 s−1, (kcat/KM)G1 = 23 μM−1s−1, (kcat)G1 = 15 s−1, (kcat/KM)G2 = 3.8 μM−1s−1, (kcat)G2 = 4.5 s−1 and kmax = 3.7 s−1. R0, vacant ribosome; R·RRF, RRF-bound ribosome; R·RRF·G, RRF and EF-G-bound ribosome; R·G, EF-G-bound ribosome. Split, the fraction of split ribosomes.(B). Simulation of the recycling reaction at 26 °C, starting from the RRF-bound post-termination complex. The distinct ribosome fractions were predicted from the reaction mechanism and its rate constants (A), and plotted as functions of time. The dashed lines indicate the sample composition at 140 ms incubation time.

Altogether in this study we obtained several cryo-EM structures of the 30S subunit, 50S subunit and the 70S ribosome complex from 3D classification of data collected with three experimental conditions: control experiment (details are in the next section), at the 140ms time point and after long (30 min) incubation. Classification of these data produced several cryo-EM structures listed with their abbreviations inTable 1. Henceforth we will refer to the structures with these abbreviations.

Table 1. List of the Cryo-EM structures obtained in this study.

In the control experiment, EF-G and IF3 were left out. Structures obtained at 140 ms are subscripted with “140”. Abbreviations: RRF, ribosome recycling factor; EF-G, elongation factor G; IF3, initiation factor 3. In the long-incubation experiment, samples are incubated first, then forced through the time-resolved apparatus for spraying. Note that all the experiments were done with the same time-resolved apparatus.

ExperimentRibosome and other components (Rot or Non-Rot)Abbreviation(Resolution/Å)
Control70S ribosome, RRF, P/E site tRNA, mRNA, (Rot)PostTC•RRFcontrol (10)
140 ms70S ribosome, RRF, P/E site tRNA, mRNA, (Rot)PostTC•RRF140(15)
70S ribosome, RRF, mRNA, (Non-Rot)NR-PostTC•RRF140 (16)
70S ribosome, RRF, P/E tRNA, EF-G, mRNA (Rot)PostTC•RRF•EF-G140 (7.4)
50S subunit, RRF, EF-G50S•RRF•EF-G140 (12)
50S subunit, RRF, EF-G, E tRNA50S•RRF•EF-G•tRNA140(16)
30S subunit, P/I tRNA, mRNA30S•tRNA140 (10)
30S subunit, IF3, mRNA30S•IF3140 (22)
Long incubation50S subunit, RRF, EF-G50S•RRF•EF-Glong (14)
50S subunit, RRF, EF-G, E tRNA50S•RRF•EF-G•tRNAlong (12)
50S subunit, RRF, E tRNA50S•RRF•tRNAlong (16)
50S subunit, E tRNA50S•tRNAlong (16)
30S subunit, IF3, mRNA30S•IF3long (10)

Control experiment confirming the stability of the RRF-bound post-termination complex

We performed a control experiment in the absence of EF-G and IF3 to validate the purity and stability of an RRF-bound post-termination complex during its passage through the mixing and reaction channel of the microfluidic device. For this experiment we employed a device with reaction time of 560 ms, having the longest reaction channel among the presently available devices. The preparation of time-resolved grid followed the procedure of Chenet al (Chen et al., 2013). We injected a mixture containing post-termination 70S ribosome complexes (2 μM) incubated with RRF (90 μM) in polymix buffer, supplemented with components for energy regeneration, into inlet 1 and only polymix buffer along with the components for energy regeneration into inlet 2 of the microfluidic device. Equal volumes of the two reaction solutions were rapidly mixed inside the mixing channel and sprayed onto the EM grid, which was immediately plunged into liquid ethane. Details of data collection and processing can be found in theSupplementary Material.

In the dataset obtained from the control experiment, no 50S or 30S subunits were observed, and only a single 70S ribosome structure class was identified with ~9,000 particles (Figure 2), which yielded a density map of the 70S ribosome in rotated conformation carrying a P/E-site tRNA and an additional mass of density in the inter-subunit space that could be readily attributed to RRF (Borovinskaya et al., 2007) (Figure 2B). Henceforth we refer to this map obtained in the control experiment as the PostTC•RRFcontrol complex (Seetable 1). The resolution of this map, 10 Å, was estimated following the “gold standard” protocol, using the FSC = 0.143 criterion (Chen et al., 2013).

Figure 2. Post-termination 70S ribosome in complex with RRF and P/E-tRNA.

Figure 2

(A) The PostTC•RRF complex. Gold, 30S subunit; blue, 50S subunit; red, RRF; and orange red, P/E-site tRNA. h, head; sh, shoulder; sp, spur. (B) Orientation and interaction of RRF and P/E site tRNA with 30S and 50S subunit, respectively.

In the PostTC•RRFcontrol complex (10 Å) obtained in the control experiment, RRF adopts the same conformation (Figure 2B) as in the structure published before (Agrawal et al., 2004;Gao et al., 2005) (EMD-1128 and EMD-1077). However, a closer analysis of the map from previous work (Agrawal et al., 2004) (EMD-1077), where no 3D classification was employed, shows that domain II actually appears in two positions (a majority density, equivalent to the presently observed one, and a minority density, the latter only seen when the density threshold was lowered in their map). In our studies domain I of RRF in the PostTC•RRFcontrol complex is seen to reach into the interface-canyon of the 50S subunit and to make contact with several parts of the 23S rRNA (Figure 2B). The elbow between the two RRF domains faces the L7/L12 stalk-base and the α-sarcin–ricin loop (SRL) region of the 50S subunit. Domain II of RRF is found in a single conformation, pointing toward the shoulder of the 30S subunit, and contacting protein S12 (Figure 2B). No density is observed to extend toward the stalk-base of the 50S subunit, even when the density threshold is reduced. We note that this conformation is quite different from that observed by Yokoyama et al. (Yokoyama et al., 2012) In their ttRRF-bound post-termination complex, although domain I of RRF binds similarly to the 50S subunit as in our structure, domain II is rotated about the long axis of domain I toward the 50S subunit (seeSupplementary Information Figure S3).

Observed structures of the 70S recycling complex at 140 ms

In the time-resolved cryo-EM experiment capturing the recycling reaction at the 140 ms time point, after mixing the RRF-bound post-termination complex with EF-G and IF3, we identified three major classes of 70S ribosome particles by 3D classification of 45K particles. The first class yields a map quite similar to the one in the first control experiment, except that the resolution is 15.5 Å. Henceforth we refer to this complex as PostTC•RRF140 (Table 1).

The second class yields an RRF-bound non-rotated 70S complex (NR-PostTC•RRF140). As compared to PostTC•RRF, this complex displays three striking differences. First, while the 70S ribosome adopts the non-rotated state in NR-PostTC•RRF140, it adopts the rotated state in PostTC•RRFcontrol (Figure 3). Related to this change, the L1 stalk of the 50S subunit is half-closed in NR-PostTC•RRF140, but closed in PostTC•RRFcontrol. Second, NR-PostTC•RRF140 contains no tRNA density, while PostTC•RRFcontrol contains P/E tRNA (Figure 3). The third difference is related to the position of RRF: in NR-PostTC•RRF140, compared to PostTC•RRFcontrol, the whole domain I of RRF is shifted toward the peptidyl-transfer center by 5 Å, and domain II makes contact with the stalk-base of the 50S subunit. Clearly, in NR-PostTC•RRF140 there is no interaction between domain II of RRF and the 30S subunit, whereas in PostTC•RRFcontrol, domain II of RRF is in contact with protein S12.

Figure 3. Interaction of RRF and tRNA in PostTC•RRFcontrol and NR-PostTC70S•RRF140 complex.

Figure 3

(A) PostTC·RRF and NR70S·RRF140 complex in top view.(B) 30S subunit of PostTC•RRF (gold) and NR70S•RRF140 (orange) in solvent and interface views. L1, L1-stalk; hd, head; pt, platform; b, body; sp, spur; sh, shoulder; bk, beak. The rotational movement is indicated by a dashed line.

The third class of 70S ribosome particles yields a complex that has both RRF and EF-G bound to the ribosome in the rotated state, and contains density for P/E tRNA (PostTC•RRF•EF-G140;Figure 4). Here domain II of EF-G make contact with the 30S subunit while domain III of EF-G appears disordered in this map, indicating residual unresolved heterogeneity of this class. Domain IV of EF-G contacts domain II of RRF, while domain V interacts with the 50S subunit. Regarding the mechanism of ribosome splitting, it is believed that it involves a movement of RRF domain II with respect to domain I upon EF-G binding (Gao et al., 2007;Gao et al., 2005). Domain II is initially (in PostTC•RRFcontrol) in contact with S12 on the small subunit. Due to the low local resolution of the factor binding site on PostTC•RRF•EF-G140, the density mass of domain II of RRF is fused with that of EF-G. Still, it is apparent that domain II is no longer in contact with S12, and the angle between domain I and II of RRF can be determined. Comparison with PostTC•RRFcontrol shows that upon binding of EF-G the angle between domains I and II of RRF decreases by approximately 20° so that domain II points toward h44 (Figure 5). By comparison, for the heterologousE. coli PostTC•ttRRF complex (Yokoyama et al., 2012), Yokoyamaet al. obtained results that are quite different. In that study, domain II is initially in contact with the stalk-base of the 50S subunit and, upon EF-G binding, a large decrease in the angle between domains I and II of RRF (75 °) takes place as domain I moves from pointing to the 50S side to the 30S side. In addition, they observe a shift of RRF by 8 Å toward the E-site tRNA.

Figure 4. Segmented map of PostTC•RRF•EF-G140 complex.

Figure 4

(A) PostTC•RRF•EF-G140 complex. Gold: 30S subunit, blue: 50S subunit, red: RRF, orange-red: P/E site tRNA, dark blue: EF-G.(B) Zoom-in views of (A) showing EF-G and RRF interaction. Density is rendered 50% transparent to show the rigid-body fitting of the EF-G and RRF structures. Five EF-G domains and two RRF domains are labeled.

Figure 5. Movement of domain II of RRF in PostTC•RRF•EF-G140 compared to its position in PostTC•RRF140.

Figure 5

The central bridge between 30S and 50S, B2a, involves h44 (yellow) and H69 (blue). S12 protein is shown in green. RRF domain II in orange shown by the fitting of pdb structure (1EH1) into its density in PostTC•RRF•EF-G140 complex. Compared to RRF domain II in PostTC•RRF140 (red), the rotational movement of domain II toward bridge B2a is shown with an arrow. The position of domain I (gray) is the same in these two complexes. h, head; sh, shoulder; sp, spur; Sb, L7/L12 stalk base. Thumbnails in lower left depict the relationship of the views presented here to the view shown infigure 2.

50S subunit complexes observed at the 140 ms time point

We found two classes of RRF- and EF-G-containing 50S subunits at 140 ms, distinguished by the presence or absence of an E-site tRNA (“50S•RRF•EF-G•tRNA140” and “50S•RRF•EF-G140”). The RRF on the 50S subunit is in the same position as observed before by our group(Gao et al., 2005). We compared the conformation of RRF in our PostTC•RRF140 with 50S•RRF•EF-G140, NR-PostTC•RRF140 and PostTC•RRF•EF-G140 (Figure 6). Domain I of RRF binds in the same position in all four complexes; in contrast, domain II in the 50S•RRF•EF-G140 complex is extensively rotated (~60°) compared to its orientation in the PostTC•RRF140 complex. Domain II of RRF in the PostTC•RRF•EF-G140 complex lies between its positions in 50S•RRF•EF-G140 and PostTC•RRF140. Domain II of RRF in NR-PostTC•RRF140 extensively interacts with the stalk base region of the 50S subunit. Detailed comparison of the conformations of RRF and EF-G in our complexes with those in published structures can be found in theSupplementary Material.

Figure 6. Domain II of RRF is in different positions in PostTC•RRFcontrol, NR-PostTC•RRF140, PostTC•RRF•EF-G140 and 50S•RRF•EF-G140.

Figure 6

(A) Comparison of domain II of RRF in PostTC•RRFcontrol and NR-PostTC•RRF140. CP, central protuberance; Sb, stalk-base; helix H69, domain I and II of RRF are indicated. The position of RRF in PostTC•RRF140 is shown in red. RRF in NR70S•RRF140 is shown in blue mesh. Rotational movement is shown by the dashed lines.(B) Comparison of domain II of RRF in PostTC•RRFcontrol and PostTC•RRF•EF-G140. RRF in PostTC•RRF•EF-G140 is shown in grey mesh.(C) Comparison of domain II of RRF in PostTC•RRFcontrol and 50S•RRF•EF-G140. RRF in 50S•RRF•EF-G140 is shown in green mesh.

In the other 50S complex, 50S•RRF•EF-G•tRNA140, we see E-site tRNA on the 50S subunit (matching the position of E-site tRNA density fromT. thermophilus orE. coli 70S ribosome) (Schmeing et al., 2003). The L1 stalk is in the half-closed conformation, as it is in the non-rotated 70S ribosome in the presence of the E-site tRNA (Réblová et al., 2012). The elbow region of the tRNA is interacting with the L1 stalk, and its CCA end is in contact with H88 of 23S rRNA. The anticodon loop of the tRNA becomes visible when the density threshold level is reduced.

30S subunit observed at the 140 ms time point

In our tRNA-bound 30S complex map (30S•tRNA, 11 Å) obtained at the 140 ms time point, a mass density for tRNA is observed (Figure 7A). Superimposition of our map with the 30S portion of the initiation complex of Allen et al. (Allen et al., 2005) (EMD-3523) locates the tRNA to the P/I position, with the tRNA elbow shifted towards the E site (Figure 7A). Thus, even in the absence of initiation factor 2 (IF2), tRNA alone on the 30S subunit occupies the P/I site. The same observation was made by Simonetti et al. for fMet-tRNAfMet in the initiation complex fromT. thermophiles (Simonetti et al., 2008). Density for mRNA density is also observed (not shownFigure 7A for simplicity). In the other 30S complex obtained by classification (30S•IF3) we find density for IF3 but not tRNA. However, the resolution of this class (22 Å) is limited because of the small number of particles (data not shown).

Figure 7. 30S subunit classes.

Figure 7

(A) Interaction of P/I-tRNA with 30S subunit at 140 ms. Yellow, 30S subunit; green, P-tRNA; orange, P/I-tRNA; red, P/E-tRNA. The mRNA density is not shown for simplicity.(B) Interaction of IF3 with 30S subunit at 30 mins. h, head; pt, platform; b, body; bk, beak; sh, shoulder; sp, spur. The mRNA density is shown in dark blue. The density attributed to IF3 is segmented out in brown and fitted with the NTD (1TIF, red) and CTD (1TIG, purple) of the IF3 structure. Panels on the left show observed densities, on the right comparison with positions of tRNA (Allen et al., 2005) and IF3 (McCutcheon et al., 1999).

Observed structures of the 30S and 50S subunits after long reaction time

To quantify the splitting reaction and obtain better resolution for the 30S•IF3 complex, we collected additional data with a reaction time of 30 minutes, by mixing the RRF-bound post-termination complex with EF-G and IF3 in a test tube. To make the results strictly comparable with those in the time-resolved experiment, the reaction product was forced through the 140 ms chip for spraying and plunging of the EM grid. As we expected, by that time all 70S ribosomes have split, and all 30S subunits are bound with IF3. With about 9,000 particles in the 30S•IF3 class, we solved the structure of this complex to 9 Å (Figure 7B). The binding location of the IF3 to 30S subunit has been controversial in the field even though studies on IF3 have been carried out with cryo-EM (Julián et al., 2011;McCutcheon et al., 1999), chemical and site directed probing(Dallas and Noller, 2001;Fabbretti et al., 2007) and crystallography on 30S subunits soaked in IF3 solution (Pioletti et al., 2001). In our 30S structure bound with IF3, we see a clear density for the C-terminal domain of IF3 at the h44 location and its N domain in the platform region (Figure 7B). Unexpectedly, we still observe mRNA density in this complex, as well (Figure 7B).

For the 50S subunit, we observed four classes. Two of these (50S•RRF•EF-Glong and 50S•RRF•EF-G•tRNAlong) match the 50S subunit classes observed at 140 ms, while the others lack EF-G and are new (50S•RRF•tRNAlong and 50S•tRNAlong). In 50S•RRF•tRNAlong, domain I of RRF is bound at the same position as in the 50S•RRF•EF-G•tRNA and 50S•RRF•EF-G complexes. Domain II of RRF is in contact with the stalk base region of the 50S subunit.

Discussion

In this study we present a snapshot of the transient structure of anE. coli post-termination ribosome on the pathway to its splitting into subunits byE. coli RRF and EF-G in the presence of GTP. The RRF- and EF-G-bound post-termination ribosome has previously been visualized only when locked by the presence of fusidic acid or with heterologous translation factors, RRF fromT. thermophilus and EF-G fromE. coli, not active in ribosome splitting. In our case the PostTC•RRF•EF-G140 complex, which is a key intermediate in the ribosome recycling process, was formed during the active splitting reaction by homologous translation factors, in the presence of GTP, with no antibiotics and without the help of any chemical intervention or mutation. The mixing/spraying method of time-resolved cryo-EM, which allows choosing reaction times in the range of tens of milliseconds to one second, was used to trap the short-lived complex on the EM grid. The complete mechanism of the ribosome recycling reaction was biochemically characterized at 37 °C in previous work and now at 20 °C in anE. coli-basedin vitro translation system within vivo-like function. These data were used to identify the complete kinetic mechanism for ribosome recycling along with its kinetic parameters and their temperature dependences. The experiments enabled us to estimate kinetic rates of ribosome splitting at 26 °C, the temperature actually prevailing in our time-resolved cryo-EM experiments, and to optimize the experimental setup for high yield of the transient ribosome complex containing both EF-G and RRF.

At a reaction temperature of 26 °C and a reaction time of 140 ms, the model predicted fractions of ribosomes in complex with EF-G and RRF or split into subunits of 55% or 28%, while the measured fractions were 20% or 40%, respectively. An explanation for this kinetic discrepancy might be that some ribosomes form the tRNA-lacking NR-PostTC•RRF complex (Table 1). This ribosome class is, we suggest, not an authentic intermediate on the ribosome splitting pathway. It was not identified by our biochemical experiments nor is it present in our kinetic model (Supplementary), which proposes that the P-site tRNA remains ribosome-bound until subunit separation (Borg et al., 2015). As can be seen from the long-time incubation experiment, this class of ribosomes will also eventually be split. The latter result is in line with a previous observation that tRNA-lacking ribosomes can be split by RRF and EF-G, albeit more slowly than their tRNA-containing counterparts (Pavlov et al., 2008).

Among the complexes identified by classification, the most interesting ones, in terms of their potential for providing new information on the recycling process, are PostTC•RRF140 and PostTC•RRF•EF-G140, interpreted as successive complexes in which the latter is a short-lived (~100 ms) intermediate. A comparison between these complexes yields information about a rotation of RRF domain II toward domain I, into a position close to inter-subunit bridge B2a, that is potentially triggered by one of three events: binding of EF-G, GTP hydrolysis on EF-G, or Pi release, or a combination thereof. While EF-G binding and GTP hydrolysis closely follow each other, Pi release may follow with some delay. It is therefore likely that the map PostTC•RRF•EF-G140 depicts a majority population of ribosomes that are in a state prior to Pi release. As to the study byYokoyama et al. (2012) (Yokoyama et al., 2012), there are major discrepancies compared with our results in both PostTC•RRF and PostTC•RRF•EF-G complexes, precluding a detailed comparison of their findings with ours. These discrepancies may originate, in part, with the construction of their complex from heterologous components. The conformational change between PostTC•RRF and PostTC•EF-G•RRF complexes indicated by the two maps suggests that EF-G assists the movement of domain II of RRF towards the bridge B2a and jointly acts with RRF to split the post-termination complex into the two subunits.

A related issue of biological importance is the disposition of deacylated tRNA and mRNA at the end of the recycling process, and the role of IF3. Our data from the 140 ms time point, showing two classes of the 30S subunit, bound either with tRNA or IF3, suggest that the post-termination deacylated tRNA remains initially with the 30S subunit and is subsequently removed by competitive binding of IF3. This conclusion is supported by the results of the long-time incubation study (Fig. 6) in which a sole class of 30S subunit, bound with IF3 is found, interpreted as the final state after all initially bound tRNAs have been displaced. Our results thus seemed to argue in favor of the model advocated by Zavialov et al. (Zavialov et al., 2005) and against the view favored by Hirakawa et al. (Hirokawa et al., 2006), according to which the tRNA leaves the post-termination complex already during the splitting reaction. On the other hand, our finding that mRNA remains with IF3 bound 30S complex in the long-time incubation study indicates that for our choice of mRNA the recycling process is not finished. It was very recently suggested to us that the scenario advocated by Zavialov et al. may be one of two possible pathways, whose selection is conditional on the strength of the interaction of the Shine-Dalgarno (SD) sequence of the mRNA and the anti-SD sequence of the 16S rRNA (B. Cooperman and A. Kaji, personal communication). In fact the mRNA used in our experiment has a strong SD sequence, and could be responsible for the selection of the observed pathway. Resolving this issue with the present method will require novel experiments with variegated strength of the SD sequence to allow detection of mRNA’s presence or absence.

Concerning the fate of the 50S subunit, its RRF- and EF-G-bound fraction is 100% at 140ms and then decreases to 79% at the long reaction time. At 140 ms, 44 % of the RRF- and EF-G-bound fraction contains and 56% lacks an E-site bound tRNA. At long reaction time the fraction of RRF-and EF-G-bound ribosomes that contains an E-site tRNA increases to 82%. We tentatively interpret this to mean that splitting of the post-termination ribosome results in an RRF- and EF-G-containing 50S subunit lacking an E-site tRNA, and that this is the dominating 50S complex also at the 140 ms reaction time. With increasing reaction time RRF, EF-G and deacylated tRNA will eventually reach the steady state for their 50S-bound and free forms that is observed at the long reaction time. By hypothesis, this would mean that the 50S fraction with E-site bound tRNA increases over time, in line with the recycling model by Zavialov et al. (Zavialov et al., 2005).

The present cryo-EM study of a very short-lived ribosome complex on the pathway to its splitting into subunits provides a proof of principle for cryo-EM visualization of short-lived, native reaction intermediates in biological processes. This, we suggest, will open the door to high-resolution structural analysis of a wide range of previously inaccessible large biological complexes in their well-defined functional contexts. We also note that time-resolved cryo-EM can be used as a very powerful experimental technique for kinetic modeling of complex biochemical processes along with precise estimation of the kinetic parameters of these models. With this technique, and by virtue of the classification methods, essentially all intermediate large molecular complexes of a reaction pathway can be identified from their high-resolution cryo-EM structures and their time evolutions can be precisely monitored by single-particle counting that is the byproduct of classification.

Currently time-resolved cryo-EM is still a somewhat cumbersome technique because of limitations to data collection yield, but we are optimistic that technological advances will soon allow the full potential of this method, with its deep integration of structural and functional approaches, to manifest in a multitude of experimental applications. It should be noted that a previous study using time-resolved cryo-EM of the back-translocation process (Fischer et al., 2010) had a time resolution in the minute range. It is the achievement of a time resolution in the more important 10 ms to 1 s range that has the potential of revolutionizing structural biology by facilitating studies of transient large molecular complexes operating in their proper biological contexts and time scales.

In most of the studies carried out either with cryo-EM (Barat et al., 2007;Gao et al., 2005;Hirashima and Kaji, 1973;Yokoyama et al., 2012) or X-ray crystallography (Selmer et al., 1999;Weixlbaumer et al., 2007;Wilson et al., 2004) to understand the ribosome splitting mechanism each complex was prepared separately using a chemical intervention. By employing time-resolved cryo-EM, in contrast, we have captured most of the transient structures that exist during ribosome splitting with a single biochemical preparation, which proves to be of advantage in terms of experimental control.

Concerning future directions of the present work on ribosome recycling, we will attempt to closely monitor the conformational dynamics, in the ribosome and in the translation factors, that lead to ribosome splitting This type of studies will have a good chance to answer the long-standing question of how EF-G and RRF interact both with each other and with the ribosome to achieve separation of the ribosomal subunits, and the role of the thermodynamic driving force provided by GTP hydrolysis in the process.

Experimental procedures

Preparation of time-resolved cryo-EM grids

Quantifoil R1.2/1.3 300 mesh Cu EM grids were carbon-coated and glow-discharged following standard procedures (Grassucci et al., 2007). The ribosome recycling reaction was performed using a mixing-spraying device (Lu et al., 2009) with an environmental chamber as previously described (Chen et al.) with a few minor alterations. During the experiment, the ambient conditions were maintained at 24 – 26 °C and 80% – 90% relative humidity. In the mixing-spraying chips equal volumes of two mixtures were injected, each at a flow rate of 3 μl per second.

Materials

Ribosomes (E. coli MRE600) and mRNA, encoding the peptide fMet-Phe-Thr, were prepared as described previously(Borg et al., 2015). His-tagged EF-G and RRF were overexpressed inE. coli and purified by nickel affinity chromatography. tRNAPhe was overexpressed in and purified fromE. coli. All experiments were performed in polymix buffer, containing 95 mM KCl, 5 mM NH4Cl, 0.5 mM CaCl2, 8 mM putrescine, 1 mM spermidine, 5 mM potassium phosphate (pH 7.5), 1 mM dithioerythritol and 5 mM Mg(OAc)2. All reaction mixtures also contained components for energy supply GTP (1 mM), ATP (1 mM), phosphoenolpyruvate (PEP, 10 mM), pyruvate kinase (PK, 50 μg/ml), myokinase (MK, 2 μg/ml).

Control experiment

A post-termination complex mixture was prepared containing 70S ribosomes (2 μM), RRF (90 μM), tRNAPhe (5 μM) and MFT mRNA (10 μM). A recycling mixture was prepared containing nothing but polymix buffer and components for energy regeneration. The two mixtures were incubated for 15 min at 37 °C and then kept on ice. They were then centrifuged for 3 min at 20,800×g before being loaded into the mixing-spraying device. Equal volumes of the two mixtures were rapidly mixed in a reaction chip with 560 ms total incubation time from mixing to freezing on the EM grid.

Time resolved cryo-EM experiment

A post-termination complex mixture was prepared containing 70S ribosomes (2 μM), RRF (90 μM), tRNAPhe (5 μM) and MFT mRNA (10 μM). A recycling mixture was prepared containing IF3 (16 μM), and EF-G (20 μM). The two mixtures were incubated for 15 min at 37 °C and then kept on ice. They were centrifuged for 3 min at 20,800×g before being loaded into the mixing-spraying device. Equal volumes of the two mixtures were rapidly mixed in a reaction chip with 140 ms total incubation time from mixing to freezing on the EM grid. In long-incubation experiment, equal volumes of the two mixtures were mixed and incubated for 25 min at 25 °C. Then the reaction mixture was split into two halves and loaded into the mixing-spraying device. The EM grid was made in the same way as in 140 ms time-resolved cryo-EM experiment. The total reaction time was roughly estimated to be 30 min (25 min incubation and 5 min preparation of EM grid with mixing-spraying device).

Data Acquisition

The grids were imaged using a FEI Tecnai Polara transmission electron microscope operated at 300 kV and at a nominal magnification of ×31,000. Data sets were collected with Leginon(Potter et al., 1999) on a K2 Summit direct electron detector (Gatan, Pleasanton, CA) with a physical pixel size of 5 μm, corresponding to 1.255 Å per pixel at the specimen in electron counting mode. The dose rate was set to eight counts per physical pixel per second. The total exposure time was 10 s. 50 frames were recorded. All images were taken with a defocus in the range of 1.5 – 3 μm.

Image Processing

For the control experiment data set, after assessment of the micrographs, 963 micrographs were selected for subsequent processing. For the 140 ms experiment and long-incubation data set, 947 and 881 micrographs were selected for subsequent processing, respectively. To correct for stage movement and beam-induced movements, all 50 frames for each micrographs were aligned (Li et al., 2013). Particle picking was done with Relion 1.3(Scheres, 2012) and manually checked.

For control experiment, all picked particles were classified into four classes. One class did not conform to the known shapes of ribosomes were rejected. Good classes were regrouped and reconstructed with auto-refinement in Relion.

For 140 ms experiment, classification was done in four steps. In a first step, it is performed with low-pass-filtered reference maps and four-times binned particle images to separate the data into 30S subunits, 50S subunits and 70S ribosomes. The next level of classification resolved conformational differences, and two classes were obtained, “non-rotated 70S” (NR 70S) and “rotated 70S” (RT 70S), based on the presence or absence of intersubunit rotation. The RT 70S class was further divided into two subclasses. The RT 70S class was further divided into two subclasses, one (PostTC•RRF140) identical (save for the difference in resolution) to the control complex (PostTC•RRFcontrol), and the other (PostTC•RRF•EF-G140). Using focused classification, by applying a soft-edged mask to the EF-G and RRF-binding region, we found no evidence of subclasses in PostTC•RRF•EF-G140. The 50S subunit class was resolved into two subclasses of RRF- and EF-G-containing 50S subunits, distinct by the presence or absence of an E-site tRNA (“50S•RRF•EF-G•tRNA140” and “50S•RRF•EF-G140”). The 30S subunit class was resolved into two subclasses, one bound with tRNA in the P/I position (“30S•tRNA140”) and the other with IF3 (“30S•IF3140”).

For long-incubation experiment, in the first step of classification, we found two classes, 50S and 30S subunits. In the next level, regrouped 30S subunits or 50S subunits were classified into subclasses. All the 30S subunit was found to be bound with IF3. For 50S subunits, four classes were found, 50S•RRF•EF-Glong, 50S•RRF•EF-G•tRNAlong, 50S•RRF•tRNAlong and 50S•tRNAlong.

Supplementary Material

supplement

HIGHLIGHTS.

  • A post-termination 70SR•RF•EF-G complex exists before the onset of recycling.

  • IF3- and tRNA- bound 30S subunit help elucidate possible recycling pathways.

  • This study shows cryo-EM is able to depict short-lived states in molecular interactions.

Acknowledgments

This work was supported by HHMI and NIH R01 GM55440 (to J.F.) and by the Swedish Research Council [2015-04682], the Knut and Alice Wallenberg Foundation, RiboCORE [KAW 2009.0251] (to M.E.) and a Lijewalchs travel stipend (Uppsala University) (to A.B.).

Footnotes

SUPPLEMENTAL INFORMATION

Supplemental Information includesSupplemental Experimental Procedures and figures and can be found with this article online at (XXX)

ACCESSION NUMBERS

The electron microscopy maps have been deposited in the EMBL-European Bioinformatics Institute EM Data Bank under accession codes EMD: 8411, 8412, 8413, 8415, 8416, 8417 and 8418 (PostTC•RRFcontrol, NR-PostTC•RRF140, PostTC•RRF•EF-G140, 50S•RRF•EF-G140, 50S•RRF•EF-G•tRNA140, 30S•tRNA140 and 30S•IF3long maps, respectively).

AUTHOR CONTRIBUTIONS

Conceptualization, Z.F., S.K., A.B., M.S., B.C., M.E. and J.F.; Formal Analysis, Z.F., S.K., A.B. and M.S.; Investigation, Z.F., S.K. and A.B.; Resources, M.E. and J.F.; Writing – Original Draft, Z.F., S.K., A.B., M.E. and J.F.; Writing – Review & Editing, Z.F., S.K., A.B., M.S., B.C., M.E. and J.F.; Visualization, Z.F., S.K. and A.B.; Supervision, R.A.G., M.E. and J.F. Funding Acquisition, Z.F., S.K., A.B., M.E. and J.F.

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

References

  1. Agrawal RK, Sharma MR, Kiel MC, Hirokawa G, Booth TM, Spahn CMT, Grassucci RA, Kaji A, Frank J. Visualization of ribosome-recycling factor on the Escherichia coli 70S ribosome: Functional implications. Proc Natl Acad Sci U S A. 2004;101:8900–8905. doi: 10.1073/pnas.0401904101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Allen GS, Zavialov A, Gursky R, Ehrenberg M, Frank J. The Cryo-EM Structure of a Translation Initiation Complex from Escherichia coli. Cell. 2005;121:703–712. doi: 10.1016/j.cell.2005.03.023. [DOI] [PubMed] [Google Scholar]
  3. Barat C, Datta PP, Raj VS, Sharma MR, Kaji H, Kaji A, Agrawal RK. Progression of the Ribosome Recycling Factor through the Ribosome Dissociates the Two Ribosomal Subunits. Mol Cell. 2007;27:250–261. doi: 10.1016/j.molcel.2007.06.005. [DOI] [PubMed] [Google Scholar]
  4. Borg A, Pavlov M, Ehrenberg M. Complete kinetic mechanism for recycling of the bacterial ribosome. RNA. 2015 doi: 10.1261/rna.053157.115. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Borovinskaya MA, Pai RD, Zhang W, Schuwirth BS, Holton JM, Hirokawa G, Kaji H, Kaji A, Cate JHD. Structural basis for aminoglycoside inhibition of bacterial ribosome recycling. Nat Struct Mol Biol. 2007;14:727–732. doi: 10.1038/nsmb1271. [DOI] [PubMed] [Google Scholar]
  6. Chen B, Kaledhonkar S, Sun M, Shen B, Lu Z, Barnard D, Lu T-M, Gonzalez Ruben L, Jr, Frank J. Structural Dynamics of Ribosome Subunit Association Studied by Mixing-Spraying Time-Resolved Cryogenic Electron Microscopy. Structure. 23:1097–1105. doi: 10.1016/j.str.2015.04.007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chen S, McMullan G, Faruqi AR, Murshudov GN, Short JM, Scheres SHW, Henderson R. High-resolution noise substitution to measure overfitting and validate resolution in 3D structure determination by single particle electron cryomicroscopy. Ultramicroscopy. 2013;135:24–35. doi: 10.1016/j.ultramic.2013.06.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Dallas A, Noller HF. Interaction of Translation Initiation Factor 3 with the 30S Ribosomal Subunit. Mol Cell. 2001;8:855–864. doi: 10.1016/s1097-2765(01)00356-2. [DOI] [PubMed] [Google Scholar]
  9. Dunkle JA, Wang L, Feldman MB, Pulk A, Chen VB, Kapral GJ, Noeske J, Richardson JS, Blanchard SC, Cate JHD. Structures of the Bacterial Ribosome in Classical and Hybrid States of tRNA Binding. Science. 2011;332:981–984. doi: 10.1126/science.1202692. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Fabbretti A, Pon CL, Hennelly SP, Hill WE, Lodmell JS, Gualerzi CO. The Real-Time Path of Translation Factor IF3 onto and off the Ribosome. Mol Cell. 2007;25:285–296. doi: 10.1016/j.molcel.2006.12.011. [DOI] [PubMed] [Google Scholar]
  11. Fischer N, Konevega AL, Wintermeyer W, Rodnina MV, Stark H. Ribosome dynamics and tRNA movement by time-resolved electron cryomicroscopy. Nature. 2010;466:329–333. doi: 10.1038/nature09206. [DOI] [PubMed] [Google Scholar]
  12. Fujiwara T, Ito K, Nakayashiki T, Nakamura Y. Amber mutations in ribosome recycling factors of Escherichia coli and Thermus thermophilus: evidence for C-terminal modulator element. FEBS Letters. 1999;447:297–302. doi: 10.1016/s0014-5793(99)00302-6. [DOI] [PubMed] [Google Scholar]
  13. Gao N, Zavialov AV, Ehrenberg M, Frank J. Specific Interaction between EF-G and RRF and Its Implication for GTP-Dependent Ribosome Splitting into Subunits. Journal of Molecular Biology. 2007;374:1345–1358. doi: 10.1016/j.jmb.2007.10.021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Gao N, Zavialov AV, Li W, Sengupta J, Valle M, Gursky RP, Ehrenberg M, Frank J. Mechanism for the Disassembly of the Posttermination Complex Inferred from Cryo-EM Studies. Mol Cell. 2005;18:663–674. doi: 10.1016/j.molcel.2005.05.005. [DOI] [PubMed] [Google Scholar]
  15. Grassucci RA, Taylor DJ, Frank J. Preparation of macromolecular complexes for cryo-electron microscopy. Nat Protocols. 2007;2:3239–3246. doi: 10.1038/nprot.2007.452. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hirashima A, Kaji A. Role of Elongation Factor G and a Protein Factor on the Release of Ribosomes from Messenger Ribonucleic Acid. Journal of Biological Chemistry. 1973;248:7580–7587. [PubMed] [Google Scholar]
  17. Hirokawa G, Demeshkina N, Iwakura N, Kaji H, Kaji A. The ribosome-recycling step: consensus or controversy? Trends BiochemSci. 2006;31:143–149. doi: 10.1016/j.tibs.2006.01.007. [DOI] [PubMed] [Google Scholar]
  18. Ito K, Fujiwara T, Toyoda T, Nakamura Y. Elongation Factor G Participates in Ribosome Disassembly by Interacting with Ribosome Recycling Factor at Their tRNA-Mimicry Domains. Mol Cell. 2002;9:1263–1272. doi: 10.1016/s1097-2765(02)00547-6. [DOI] [PubMed] [Google Scholar]
  19. Julián P, Milon P, Agirrezabala X, Lasso G, Gil D, Rodnina MV, Valle M. The Cryo-EM structure of a complete 30S translation initiation complex from Escherichia coli. PLoS Biol. 2011;9:e1001095. doi: 10.1371/journal.pbio.1001095. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Karimi R, Pavlov MY, Buckingham RH, Ehrenberg M. Novel Roles for Classical Factors at the Interface between Translation Termination and Initiation. Mol Cell. 1999;3:601–609. doi: 10.1016/s1097-2765(00)80353-6. [DOI] [PubMed] [Google Scholar]
  21. Kim KK, Min K, Suh SW. Crystal structure of the ribosome recycling factor from Escherichia coli. The EMBO Journal. 2000;19:2362–2370. doi: 10.1093/emboj/19.10.2362. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Lancaster L, Kiel MC, Kaji A, Noller HF. Orientation of Ribosome Recycling Factor in the Ribosome from Directed Hydroxyl Radical Probing. Cell. 2002;111:129–140. doi: 10.1016/s0092-8674(02)00938-8. [DOI] [PubMed] [Google Scholar]
  23. Lu Z, Shaikh TR, Barnard D, Meng X, Mohamed H, Yassin A, Mannella CA, Agrawal RK, Lu TM, Wagenknecht T. Monolithic microfluidic mixing–spraying devices for time-resolved cryo-electron microscopy. J Struct Biol. 2009;168:388–395. doi: 10.1016/j.jsb.2009.08.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. McCutcheon JP, Agrawal RK, Philips SM, Grassucci RA, Gerchman SE, Clemons WM, Ramakrishnan V, Frank J. Location of translational initiation factor IF3 on the small ribosomal subunit. Proceedings of the National Academy of Sciences. 1999;96:4301–4306. doi: 10.1073/pnas.96.8.4301. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Nakano H, Yoshida T, Uchiyama S, Kawachi M, Matsuo H, Kato T, Ohshima A, Yamaichi Y, Honda T, Kato H, et al. Structure and Binding Mode of a Ribosome Recycling Factor (RRF) from Mesophilic Bacterium. Journal of Biological Chemistry. 2003;278:3427–3436. doi: 10.1074/jbc.M208098200. [DOI] [PubMed] [Google Scholar]
  26. Pavlov MY, Antoun A, Lovmar M, Ehrenberg M. Complementary roles of initiation factor 1 and ribosome recycling factor in 70S ribosome splitting. The EMBO Journal. 2008;27:1706–1717. doi: 10.1038/emboj.2008.99. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Pioletti M, Schlünzen F, Harms J, Zarivach R, Glühmann M, Avila H, Bashan A, Bartels H, Auerbach T, Jacobi C, et al. Crystal structures of complexes of the small ribosomal subunit with tetracycline, edeine and IF3. The EMBO Journal. 2001;20:1829–1839. doi: 10.1093/emboj/20.8.1829. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Potter CS, Chu H, Frey B, Green C, Kisseberth N, Madden TJ, Miller KL, Nahrstedt K, Pulokas J, Reilein A, et al. Leginon: a system for fully automated acquisition of 1000 electron micrographs a day. Ultramicroscopy. 1999;77:153–161. doi: 10.1016/s0304-3991(99)00043-1. [DOI] [PubMed] [Google Scholar]
  29. Réblová K, Šponer J, Lankaš F. Structure and mechanical properties of the ribosomal L1 stalk three-way junction. Nucleic Acids Research. 2012;40:6290–6303. doi: 10.1093/nar/gks258. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Raj VS, Kaji H, Kaji A. Interaction of RRF and EF-G from E. coli and T. thermophilus with ribosomes from both origins—insight into the mechanism of the ribosome recycling step. RNA. 2005;11:275–284. doi: 10.1261/rna.7201805. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Ramakrishnan V. Ribosome structure and the mechanism of translation. Cell. 2002;108:557–572. doi: 10.1016/s0092-8674(02)00619-0. [DOI] [PubMed] [Google Scholar]
  32. Savelsbergh A, Rodnina MV, Wintermeyer W. Distinct functions of elongation factor G in ribosome recycling and translocation. RNA. 2009;15:772–780. doi: 10.1261/rna.1592509. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Scheres SHW. RELION: Implementation of a Bayesian approach to cryo-EM structure determination. J Struct Biol. 2012;180:519–530. doi: 10.1016/j.jsb.2012.09.006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Schmeing TM, Moore PB, Steitz TA. Structures of deacylated tRNA mimics bound to the E site of the large ribosomal subunit. RNA. 2003;9:1345–1352. doi: 10.1261/rna.5120503. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Selmer M, Al-Karadaghi S, Hirokawa G, Kaji A, Liljas A. Crystal Structure of Thermotoga maritima Ribosome Recycling Factor: A tRNA Mimic. Science. 1999;286:2349–2352. doi: 10.1126/science.286.5448.2349. [DOI] [PubMed] [Google Scholar]
  36. Shaikh TR, Yassin AS, Lu Z, Barnard D, Meng X, Lu TM, Wagenknecht T, Agrawal RK. Initial bridges between two ribosomal subunits are formed within 9.4 milliseconds, as studied by time-resolved cryo-EM. Proceedings of the National Academy of Sciences. 2014;111:9822–9827. doi: 10.1073/pnas.1406744111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Simonetti A, Marzi S, Myasnikov AG, Fabbretti A, Yusupov M, Gualerzi CO, Klaholz BP. Structure of the 30S translation initiation complex. Nature. 2008;455:416–420. doi: 10.1038/nature07192. [DOI] [PubMed] [Google Scholar]
  38. Toyoda T, Tin OF, Ito K, Fujiwara T, Kumasaka T, Yamamoto M, Garber MB, Nakamura Y. Crystal structure combined with genetic analysis of the Thermus thermophilus ribosome recycling factor shows that a flexible hinge may act as a functional switch. RNA. 2000;6:1432–1444. doi: 10.1017/s1355838200001060. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Weixlbaumer A, Petry S, Dunham CM, Selmer M, Kelley AC, Ramakrishnan V. Crystal structure of the ribosome recycling factor bound to the ribosome. Nat Struct Mol Biol. 2007;14:733–737. doi: 10.1038/nsmb1282. [DOI] [PubMed] [Google Scholar]
  40. Wilson DN, Schluenzen F, Harms JM, Yoshida T, Ohkubo T, Albrecht R, Buerger J, Kobayashi Y, Fucini P. X-ray crystallography study on ribosome recycling: the mechanism of binding and action of RRF on the 50S ribosomal subunit. The EMBO Journal. 2004;24:251–260. doi: 10.1038/sj.emboj.7600525. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Yokoyama T, Shaikh TR, Iwakura N, Kaji H, Kaji A, Agrawal RK. Structural insights into initial and intermediate steps of the ribosome-recycling process. The EMBO Journal. 2012;31:1836–1846. doi: 10.1038/emboj.2012.22. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Yoshida T, Oka S, Uchiyama S, Nakano H, Kawasaki T, Ohkubo T, Kobayashi Y. Characteristic Domain Motion in the Ribosome Recycling Factor Revealed by 15N NMR Relaxation Experiments and Molecular Dynamics Simulations. Biochemistry. 2003;42:4101–4107. doi: 10.1021/bi027191y. [DOI] [PubMed] [Google Scholar]
  43. Yoshida T, Uchiyama S, Nakano H, Kashimori H, Kijima H, Ohshima T, Saihara Y, Ishino T, Shimahara H, Yoshida T, et al. Solution Structure of the Ribosome Recycling Factor from Aquifex aeolicus‡. Biochemistry. 2001;40:2387–2396. doi: 10.1021/bi002474g. [DOI] [PubMed] [Google Scholar]
  44. Zavialov AV, Hauryliuk VV, Ehrenberg M. Splitting of the posttermination ribosome into subunits by the concerted action of RRF and EF-G. Mol Cell. 2005;18:675–686. doi: 10.1016/j.molcel.2005.05.016. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

supplement

ACTIONS

RESOURCES

[8]ページ先頭
©2009-2025 Movatter.jp