Mechanism of Translation Termination: RF1 Dissociation Follows

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Mechanism of Translation Termination: RF1 Dissociation follows RF3 Dissociation from the Ribosome Xinying Shi, and Simpson Joseph Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.6b00921 • Publication Date (Web): 25 Oct 2016 Downloaded from http://pubs.acs.org on October 28, 2016

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Biochemistry

Mechanism of Translation Termination: RF1 Dissociation follows RF3 Dissociation from the Ribosome

Xinying Shi and Simpson Joseph

Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0314.

Running Title: Timing of RF1 and RF3 dissociation

Correspondence to: Simpson Joseph 4102 Urey Hall, Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0314.

Phone: (858) 822-2957 Fax: (858) 534-7042 E-mail: [email protected]

1 ACS Paragon Plus Environment

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Abbreviations RF1, release factor 1; RF2, Release factor 2; RF3, release factor 3; tRNA, transfer RNA; mRNA, messenger RNA; FRET, Förster resonance energy transfer


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Abstract

Release factors 1 and 2 (RF1 and RF2) bind to ribosomes that have a stop codon in the A site and catalyze the release of the newly synthesized protein. Following peptide release, the dissociation of RF1 and RF2 from the ribosome is accelerated by release factor 3 (RF3). The mechanism for RF3-promoted dissociation of RF1/RF2 is unclear. It was previously proposed that RF3 hydrolyzes GTP and dissociates from the ribosome after RF1 dissociation. Here we monitored directly the dissociation kinetics of RF1 and RF3 using Förster resonance energy transfer (FRET) based assays. In contrast to the previous model, our data show that RF3 hydrolyzes GTP and dissociates from the ribosome before RF1 dissociation. We propose that RF3 stabilizes the ratcheted state of the ribosome, which consequently accelerates the dissociation of RF1 and RF2.


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During the termination step of bacterial protein synthesis, release factors 1 and 2 (RF1 and RF2) bind to ribosomes that display a stop codon in the ribosomal A site to promote the hydrolytic release of the polypeptide from the tRNA in the P site [reviewed in

1, 2

]. RF1 recognizes the stop codons UAA and UAG, whereas RF2 recognizes the

stop codons UAA and UGA 3. RF1/RF2, bound to the ribosomal A site, span the distance between the decoding center of the 30S small ribosomal subunit and the peptidyl transferase center of the 50S large ribosomal subunit 4-7. Domain II of RF1/RF2 interact with the stop codon in the decoding center, and the universally conserved residues GGQ in domain III reaches into the peptidyl transferase center to promote peptidyl-tRNA hydrolysis

6, 7

.

After catalyzing peptidyl-tRNA hydrolysis, RF1/RF2 remain tightly

bound to the ribosome, and their dissociation is promoted by release factor 3 (RF3) 8. RF3 is a guanosine triphosphatase (GTPase), but the role of GTP hydrolysis and the mechanism of RF3-promoted dissociation of RF1 and RF2 are not fully understood. A previous study showed that free RF3 has a 500-fold higher affinity for GDP compared to GTP suggesting that RF3 is bound to GDP when it binds to the post-termination complex in vivo (post-termination complex is defined as ribosome with RF1 or RF2 in the A site after the peptidyl-tRNA hydrolysis reaction) 9. The post-termination complex was shown to accelerate the dissociation of GDP from RF3, which results in the binding of GTP to RF3 9. Previous studies also showed that RF3GTP decreases the “recycling time” of RF1 and RF2 when they are present in sub-stoichiometric amounts relative to the ribosomal complex 8, 9. Lastly, structural studies have shown that RF3GDPNP binds to the ribosome and induces a counter-clockwise ratchet-like rotation of the small ribosomal subunit relative to the large ribosomal subunit

10

.

Based on these data,


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Ehrenberg and co-workers proposed a model for RF3-mediated acceleration of RF1 and RF2 dissociation from the ribosome

9, 10

. According to this model, RF3, in the GDP

form, binds with low affinity to the post-termination complex containing either RF1 or RF2. The post-termination complex triggers the dissociation of GDP from RF3. GTP then binds rapidly to apo-RF3 to form a high-affinity RF3GTP complex with the ribosome. Binding of GTP to RF3 induces conformational changes both in RF3 and the ribosome. Apo-RF3 changes from a semi-closed to an open conformation when it binds GTP and the ribosome changes from the classic unratcheted to the ratcheted state. These conformational changes by the ribosome destabilize the interaction of RF1/RF2 with the post-termination complex leading to the release of RF1/RF2. Finally, RF3 hydrolyzes GTP to switch back to its low affinity GDP form and rapidly dissociates from the ribosome bringing the ribosome back to the unratcheted state. Recent studies have shown that RF3 binds GDP and GTP with comparable affinities suggesting that RF3 exist predominantly in the GTP bound form in the cell due to the higher GTP concentration than GDP 11, 12. This finding has led to a revised model, in which RF3GTP directly binds to the post-termination complex followed by ratcheting of the ribosome and the release of RF1/RF2. RF3 then hydrolyses GTP and RF3GDP dissociates from the ribosome. However, key steps of the previous model, such as the dissociation of RF1 and RF3, have not been directly observed but were inferred from steady-state experiments

9, 10

. Here, we used Förster resonance energy transfer (FRET)

based assays to directly monitor the dissociation of RF1 and RF3 from the posttermination complex. Additionally, we used GTPase-deficient RF3 mutants to analyze the role of GTP hydrolysis in RF1 dissociation. Surprisingly, our data show that the


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dissociation of RF3 from the ribosome precedes RF1 dissociation. These data suggest that RF3 stabilizes the ratcheted state of the ribosome, which consequently accelerates the dissociation of RF1 and RF2.


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EXPERIMENTAL PROCEDURES Site-directed mutagenesis of RF1 and RF3 The E. coli wild type RF1 gene cloned into pET24b plasmid was described previously 13, 14

. The single cysteine RF1 mutant was made by changing the three native cysteines to

serine and a new cysteine was introduced at position 192 (Ser192Cys) by QuickChange site-directed mutagenesis (Stratagene). RF1 (GAQ) mutant was made by changing the glutamine at position 234 to alanine by site-directed mutagenesis using the wild type RF1 plasmid as the template. Similarly, we used site-directed mutagenesis to make RF3 mutants Thr27Ala, Ser69Ala, and His92Ala in plasmid pTYB1 (New England Biolabs). RF3 has six native cysteines.

To make the single cysteine RF3 mutant for FRET

experiments, we changed Cys at five positions to serine (Cys82Ser, Cys108Ser, Cys109Ser, Cys166Ser, and Cys175Ser) but kept the native Cys at position 476 for attaching fluorescein. The gene coding for the single cysteine RF3 was sub-cloned into the pMCSG9 vector 15.

Purification of ribosome, tRNA, mRNA, RF1 and RF3 Tight-couple 70S ribosomes were purified from E. coli strain MRE600 as described

16

.

tRNAfMet were prepared as described 17. Synthetic mRNA (21 nucleotides) having a start codon (AUG) followed by a stop codon (UAA) and a 3’-amino-modifier C3 linker was purchased from Dharmacon

14

. All proteins were overexpressed in E. coli BL21(DE3).

Wild type RF1, RF1 (Ser192Cys) mutant and RF1 (GAQ) mutant proteins were purified as described before and stored at -20 °C

14

. RF3 (Thr27Ala), RF3 (Ser69Ala) RF3

(His92Ala) mutants and wild type RF3 were purified by following the manufacturer’s 7 ACS Paragon Plus Environment

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instruction (New England Biolabs) and stored in buffer (300 mM NaCl, 10 µM GDP, 1 mM DTT, 50 mM Tris-HCl, pH 8.0) at -80 °C. The single cysteine RF3 was first purified by Ni-NTA affinity chromatography.

After cleavage by TEV protease

overnight, protein was passed through a Ni-NTA affinity column to remove his-tagged maltose binding protein and his-tagged TEV. RF3 was further purified by size exclusion chromatography with a Superdex-200 (16/600) column and stored in buffer (250 mM KCl, 10 mM MgCl2, 10% glycerol, 10 µM GDP, 50 mM K-Hepes, pH 8.0) at -20 °C. For the mantGTP binding studies, RF3 was stored in buffer without GDP.

Protein and mRNA labeling RF1 and RF3 containing a single cysteine were labeled with 5-iodoacetamido-fluorescein (IAF; Invitrogen). RF1 was buffer exchanged into labeling buffer (100 mM KCl, 50 mM K-Hepes, pH 8.0) before the labeling reaction. RF3 was labeled in storage buffer. Labeling reactions were performed with 10-fold excess IAF for 2 hours at room temperature. Excess dye was removed by passing through Bio-Spin P-6 size exclusion column (Bio-Rad) and dialyzed against storage buffer overnight. Labeled proteins were flash frozen in liquid nitrogen and stored at -80 °C. mRNA was labeled with NHSRhodamine (Thermo Scientific) as described before 18.

Binding of mantGTP to RF3

Binding of mantGTP to wild type and mutant RF3 were monitored by the increase in the emission intensity of mantGTP. All experiments were done in buffer A (50 mM Tris-


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HCl pH 7.0, 100 mM NH4Cl, 10 mM MgCl2 and 1 mM DTT). Three reactions were analyzed for the wild type RF3 and each RF3 mutants: (i) mantGTP (250 nM), (ii) mantGTP (250 nM) plus wild type or mutant RF3 (500 nM), and (iii) mantGTP (250 nM) plus wild type or mutant RF3 (500 nM) plus 5 µM GTP (“chase”). As a control, we prepared an identical set with RF1. The reactions were incubated at 25 °C for 30 minutes then 200 µl of each reaction was excited at 355 nm (5 nm excitation band pass) and the fluorescence emission spectra from 425 nm to 500 nm (5 nm emission band pass) was monitored with a fluorometer (Jasco FP-8500). The % change in fluorescence intensity at 443 nm was calculated using the mantGTP only sample as the baseline.

The

experiment was done twice to calculate the mean ± SD.

Assay for RF1 and RF3 binding to ribosome Ribosome (0.7 µM) was activated at 42 °C for 10 minutes in the buffer A, followed by incubation at 37 °C for 10 minutes. mRNA (1.4 µM) and deacylated tRNAfMet (1.4 µM) were added to the ribosome and the complex was incubated at 37 °C for 30 minutes. Next, either buffer (no RF1 samples) or RF1 (2.8 µM) were added and complexes were incubated at 37 °C for 10 minutes followed by the addition of RF3 (2.8 µM) and 1 mM GDP, GDPCP, GDPNP, or GTP and incubated at 37 °C for 10 minutes. Final volume of each reaction was 30 µl. Sucrose cushions were prepared by adding 200 µl of sucrose solution (1.1 M sucrose, 20 mM Tris-HCl pH 7.5, 100 mM NH4Cl, 10 mM MgCl2 and 1 mM DTT) to centrifuge tubes (Beckman catalog# 343775) and the sucrose cushions were cooled to 4 °C. Each ribosome-binding reaction was carefully layered on top of a sucrose cushion and the tubes were centrifuged at 38,000 rpm for 2 hours at 4 °C in a Beckman


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Type 42.2 Ti rotor. After centrifugation, the sucrose solution was carefully removed from the tubes. Each ribosomal pellet was dissolved in 12 µl of buffer A then mixed with 3 µl of 5X loading buffer and separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The gels were stained with coomassie blue then scanned with a digital scanner and quantified using Quantity One (Bio-Rad). The ribosomal protein S2 band was used for normalization of RF1 and RF3 bands.

GTP hydrolysis by RF3 Post-termination complex (PostTC) were formed in buffer A by combining heat-activated ribosome (0.2 µM), mRNA (0.4 µM) and deacylated tRNAfMet (0.4 µM) [or deacylated tRNAPhe (0.4 µM) for some experiments] and incubating at 37 °C for 30 minutes. Then, RF1 (0.4 µM) was added to the reaction and the incubation was continued for another 10 minutes. The PostTC (0.2 CM) was mixed with RF3 (0-50 µM) and GTP (1 mM final concentration and trace amounts (0.02 µCi/µl) of [γ32P]-GTP (specific activity = 6000 Ci/mmol) in buffer A. The reaction was incubated at room temperature, and 3µl aliquots were withdrawn at each time point and quenched with 1 µl of 5% SDS. Samples were analyzed by thin layer chromatography (PEI cellulose) developed in 0.5 M KH2PO4 (pH 3.5). The extent of GTP hydrolysis was quantified with a phosphorimager. To calculate kGTP, the initial velocity of GTP hydrolysis derived from the initial slopes versus RF3 concentration was plotted and fit to the Michaelis-Menten equation 19. Experiments were repeated three times to calculate standard deviations. GTP hydrolysis experiments were performed both with wild type RF3 and the single cysteine RF3 mutant and they gave similar rates indicating that that the single cysteine RF3 is fully functional.


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Dissociation Complex formation For RF1 dissociation assay, PostTC were formed in buffer A by combining ribosome (0.2 µM), rhodamine labeled mRNA (0.4 µM) and deacylated tRNAfMet (0.4 µM) and incubating at 37 °C for 30 minutes. Then, fluorescein labeled RF1 (0.2 µM) was added to the reaction and the incubation was continued for another 10 minutes. Separately, RF1 chase reaction mix was prepared by combining RF1 (2 µM), RF3 (2 µM) and 1 mM of the appropriate guanine nucleotide.

For RF3 dissociation assay, experiments were performed in buffer B (50 mM Tris-HCl pH 7.5, 100 mM NH4Cl, 10 mM MgCl2, 0.015% nikkol, and 1 mM DTT). Ribosome (0.5 µM), rhodamine labeled mRNA (0.9 µM) and tRNAfMet (1 µM) were combined and incubated at 37 °C for 30 minutes. Then, RF1 (2.5µM) was added and the incubation was continued for 10 minutes to form the PostTC. Finally, fluorescein labeled apo-RF3 (0.2 µM) was added to the PostTC and the reaction was incubated for 10 minutes at 37 °C. Separately, RF3 chase reaction mix was prepared by combining unlabeled RF3 (2 µM) and 1 mM of the appropriate guanine nucleotide.

Steady state FRET measurements FRET was measured with a photon-counting instrument (FluoroMax-3, J.Y. Horiba Inc, USA). The temperature of the sample was maintained constant at 25 °C. 200 µl aliquot of dissociation complex, prepared as described above, was excited at 470 nm wavelength


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(2 nm excitation band pass) and the emission spectrum from 480nm to 660 nm wavelength was recorded (2 nm emission band pass, path length 3 mm).

Dissociation kinetics of RF1 and RF3 from ribosomes Rapid kinetic experiments were performed on a stopped-flow instrument (Bio-Logic Instruments, France). For the RF1 dissociation assay, syringe 1 contained 0.5 ml of 70SRF1-FL post-termination complex (0.2 µM) and syringe 2 contained 0.5 ml of RF1 chase reaction mix (see above). For the RF3 dissociation assay, syringe 1 contained 0.5 ml of 70SRF1apoRF3-FL complex (0.2 µM) and syringe 2 contained 0.5 ml of RF3 chase reaction mix (see above). Dissociation was initiated by rapidly mixing 80 µl of sample from syringe 1 with 80 µl of sample from syringe 2 at 25 °C. Samples were excited at 470 nm (excitation band pass 10 nm, path length 2 mm) and fluorescence emission was measured after passing a 525 nm long-pass filter (HQ525/25M, 25 mm diameter, Chroma Technology Corp. USA). The change in fluorescein fluorescence intensity over time was measured. For RF1 dissociation experiments, the stopped-flow data was collected for 16 sec with RF3GTP, 40 sec with RF3 (His92Ala) mutant with GTP, 80 sec with RF3GDPNP, 400 sec for both RF3GDP and control reaction without RF3. For RF3 dissociation experiments, the stopped-flow data was collected for 16 sec with the different guanine nucleotides. Each experiment consisted typically of four shots. The data from four shots were averaged and analyzed to determine the rate constant of dissociation. Data (8000 data points per shot) were evaluated by fitting to the single exponential equation Y = A1 exp(-k1x) for the RF1 dissociation kinetics and to the double exponential equation Y = A1 exp(-k1x) + A2 exp(-k2x) for the RF3 dissociation kinetics


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using GraphPad Prism software. All experiments were independently repeated three times to calculate standard deviations. Since the dissociation of RF3 showed biphasic kinetics, we analyzed the data by nonlinear regression using the DynaFit program

20

in

terms of Scheme 1:

Step 1 is a conformational change of the PostTCRF3* complex with the rate constants k1

and k+1 and step 2 is the dissociation of RF3 from the PostTC with the rate constant k-2.

The second step is essentially irreversible because we used a large excess of unlabeled RF3 as a trap to bind to the PostTC. To avoid picking local minima we tested a wide range of values for k-1, k+1, and k-2 using DynaFit.

Statistical criteria and visual

inspection of the residual distribution plots were used to obtain the best fits. The RF3 dissociation experiment for each nucleotide was repeated at least twice and the kinetic traces were independently fitted.


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Results

RF3 binds to the ribosome in the GTP form

To understand the role of GTP binding to RF3 and the hydrolysis of GTP in promoting the dissociation of RF1 from the ribosome, we analyzed three different RF3 mutants: Thr27Ala, Ser69Ala, and His92Ala. Previous structural data indicated that a conserved histidine (His84 in EF-Tu, which is functionally equivalent to His92 in RF3) in the active site, acts as a general base in catalyzing GTP hydrolysis in all translational GTPases

21

. However, the role of this conserved histidine in catalyzing GTP hydrolysis

has been debated in the literature

22, 23

.

Moreover, a recent crystal structure of

RF3GDPNP bound to the ribosome showed that His92 is oriented away from the γphosphate suggesting that it is not critical for GTP hydrolysis

24

. Instead, in the crystal

structure of RF3GDPNP bound to the ribosome, the β- and γ-phosphates of GDPNP interact with a magnesium ion that is coordinated by Thr27 and Ser69 of RF3. The magnesium ion may play a critical role in GTP hydrolysis by stabilizing the developing negative charge on the β-γ bridge oxygen. Because of conflicting structural data, the precise mechanism of GTP hydrolysis by RF3 remains unclear.

Furthermore,

biochemical data analyzing the contribution of His92, Thr27 and Ser69 in RF3 to GTP hydrolysis is also lacking. We first analyzed the binding of GTP to the three RF3 mutants (His92Ala, Ser69Ala, and Thr27Ala). Previous studies have shown that the binding of mantGTP, a fluorescent analog of GTP, to RF3 increases its fluorescence emission intensity

11, 12

.


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MantGTP with wild type or RF3 mutants showed the expected increase in fluorescence intensity (Figure 1). Additionally, when excess GTP (chase) was added to compete with mantGTP for binding to RF3, the fluorescence intensity decreased. These results show that wild type and the three RF3 mutants can bind GTP. We next analyzed the binding of the three RF3 mutants to ribosomes in the absence or presence of RF1. Ribosome complexes had an mRNA with a UAA stop codon in the A site and a tRNAfMet in the P site. Binding of RF3 to the ribosome was monitored by ultracentrifugation through a sucrose cushion. RF3 bound to the ribosome co-sediments with the ribosome to the bottom of the tube, whereas free RF3 remain at the top of the sucrose cushion.

The ribosomeRF3 pellet was analyzed by SDS-

polyacrylamide gel electrophoresis.

As expected from previous studies, wild type

RF3GDPCP and RF3GDPNP bound to the ribosome both in the absence and presence of RF1 (Figure 2A and Figure S1)

10-12, 24, 25

. The presence of RF1 slightly reduced the

binding of wild type RF3 to the ribosome (Figure 2B). RF3 (His92Ala)GDPCP also bound to the ribosome in the absence and presence of RF1.

In contrast, RF3

(Thr27Ala)GDPCP and RF3 (Ser69Ala)GDPCP were not retained by the ribosome in the absence of RF1 but bound to a reduced extent in the presence of RF1 suggesting that these two RF3 mutants have lower binding affinity for the ribosome. In the presence of GTP, wild type RF3 were retained poorly by the ribosome because RF3 hydrolyzes GTP to GDP and RF3GDP rapidly dissociates from the ribosome.

Interestingly, RF3 (His92Ala)GTP bound stably to the ribosome in the

absence or presence of RF1 (Figure 2A and 2B), which is consistent with the idea that His92 of RF3 is critical for GTP hydrolysis. The RF3 (His92Ala) mutant binds GTP but


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is incapable of catalyzing GTP hydrolysis, therefore, remains bound to the ribosome. In contrast, RF3 (Ser69Ala)GTP and RF3 (Thr27Ala)GTP were not retained by the ribosome with or without RF1. In the absence of any nucleotide, wild type RF3, RF3 (Ser69Ala) and RF3 (Thr27Ala) bound to ribosomes only in the presence of RF1. This is consistent with a recent report showing that apo-RF3 binds stably to ribosomes in the presence of RF1

26

. However, apo-RF3 (His92Ala) was not retained by the ribosome

even in the presence of RF1 (Figure 2A and 2B). Thus, RF3 (His92Ala) showed distinct binding properties; in the presence of GDP, GTP, or GDPCP it bound to ribosome to varying degrees but in the absence of guanine nucleotides it was retained poorly by the ribosome suggesting that the interaction of the protein with the ribosome is not stable without a guanine nucleotide.

GTP hydrolysis by RF3

Previous studies have investigated the kinetics of GTP hydrolysis by ribosomebound RF3 as a function of RF1/RF2 concentration or GTP concentration 9, 12. We were concerned that the multiple turnover rate of GTP hydrolysis may be affected by the weaker binding affinity of the RF3 mutants to the PostTC. Therefore, we determined the rate of GTP hydrolysis using a fixed low concentration of post-termination complex (PostTC) and saturating GTP concentration while varying the concentration of RF3. The PostTC consists of ribosomes programmed with an mRNA that has a stop codon in the A site, tRNAfMet in the P site and RF1 in the A site. Time course of GTP hydrolysis at increasing concentrations of RF3 was used to determine the maximum rate of GTP


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hydrolysis (Figure 3). The steady state rate of GTP hydrolysis by wild type RF3 is similar to values published previously (kGTP = 0.52 ± 0.01 s-1) 9, 12. Interestingly, all three RF3 mutants were inactive in GTP hydrolysis indicating that Thr27, Ser69 and His92 are essential for GTP hydrolysis. To investigate whether tRNAfMet in the P site may affect the rate of GTP hydrolysis we formed PostTC with tRNAPhe in the P site and RF1 in the A site. The rate of GTP hydrolysis by wild type RF3 increased slightly with tRNAPhe in the P site (kGTP = 1.05 ± 0.05 s-1) (Figure 3B), justifying the use of either tRNAs for studying the function of RF3 as was reported previously 12.

Kinetics of RF3-dependent RF1 dissociation from the ribosome

To determine the dissociation rate constant of RF1 from the ribosome, we used a FRET assay. We used the fluorescent dyes rhodamine and fluorescein as the FRET acceptor and donor, respectively. We attached rhodamine to the 3’-end of the mRNA at position +9 and fluorescein to Cys192 in RF1. When the PostTC formed with unlabeled mRNA and fluorescein-labeled RF1 was excited at 480 nm, it showed the typical fluorescence emission spectrum of fluorescein with a peak at 518 nm (Figure 4A). The PostTC formed with rhodamine-labeled mRNA and unlabeled RF1 showed very little fluorescence emission from rhodamine when excited at 480 nm. These two control reactions show that the background fluorescence emission by rhodamine is low in the absence of FRET. Importantly, the PostTC formed with rhodamine-labeled mRNA and fluorescein-labeled RF1 showed a significant decrease (≈30%) in the fluorescence


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emission of fluorescein and an increase in the fluorescence emission of rhodamine, which is characteristic of FRET (Figure 4A). We used the increase in the fluorescence emission intensity of fluorescein to monitor the rate of RF3-dependent dissociation of RF1 from the ribosome using a stopped-flow instrument.

In these stopped-flow experiments, the PostTC having

rhodamine-labeled mRNA and fluorescein-labeled RF1, was rapidly mixed with a 10fold excess of unlabeled RF1 (works as chase to prevent the rebinding of labeled RF1), 10-fold excess of RF3 and a 500 µM final concentration of GDP, GTP, or GDPNP. The results showed that the wild type RF3GTP accelerated the dissociation of RF1 by 40fold compared to RF3GDP or control without RF3 (koff = 0.38 ± 0.04 s-1 with GTP, 0.01 ± 0.01 s-1 with GDP, and 0.01 ± 0.01 s-1 without RF3) (Figure 4B and Table 1). Interestingly, RF3 (His92Ala)GTP (koff = 0.12 ± 0.04 s-1) and wild type RF3GDPNP (koff = 0.08 ± 0.02 s-1) also stimulated the dissociation of RF1 by 12- and 8-fold, respectively. These results indicate that the GTP-bound conformation of RF3 increases the dissociation of RF1 from the ribosome.

However, the rate constant of RF1

dissociation is ≈3-fold higher with wild type RF3 and GTP suggesting that GTP hydrolysis is important for the rapid release of RF1 from the termination complex. Consistent with recently published data 26, our binding studies showed that in the presence of RF1, apo-RF3 bound efficiently to the PostTC (Figure 2). To investigate the mechanism of RF1 dissociation, we monitored the rate of RF1 dissociation from the apoRF3 termination complex with the FRET assay. Apo-RF3 decreased the dissociation rate constant of RF1 from the termination complex by ≈10-fold compared to RF3GDP or even the spontaneous dissociation of RF1 (Figure 4C), and by >400-fold compared to


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RF3GTP (Table 1). In agreement with these results, addition of GDP or GTP to the apo-RF3 termination complex greatly increased the dissociation rate constant of RF1 indicating that apo-RF3 can bind GDP/GTP and trigger RF1 release (Figure 4D). The ability to make a stable termination complex having both RF1 and apo-RF3 that responds to the addition of GDP/GTP makes it possible to determine the dissociation rate constant of RF3 (described below).

The 70SRF1apoRF3 complex is relevant for kinetic

analysis because it is an intermediate in the previous model for RF3-dependent dissociation of RF1/RF2 9, 10, 26.

Kinetics of RF3 dissociation from the ribosome

To directly monitor the dissociation of RF3 from the termination complex, we used a FRET assay with rhodamine-labeled mRNA (mRNA-RH) and fluorescein-labeled RF3 (RF3-FL). Apo-RF3 was bound to the PostTC consisting of the ribosome with tRNAfMet in the P site, and RF1 in the A site. To increase the FRET signal we used a 2.5fold molar excess of PostTC over apo-RF3 so that there was very little unbound RF3. Fluorescence emission scans of PostTC having both mRNA-RH and apo-RF3-FL showed a decrease in fluorescein fluorescence intensity (≈518 nm) and an increase in rhodamine fluorescence intensity (≈585 nm) compared to control complexes (Figure 5A). This result shows that the two dyes are sufficiently close to each other in the PostTCapo-RF3 complex for FRET to occur.

As expected, the dissociation of RF3-FL from the

PostTCapo-RF3 complex by the addition of guanine nucleotide resulted in an increase


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in the emission intensity of fluorescein (Figure S2), which was used to monitor the reaction time course. We first analyzed the dissociation kinetics of apo-RF3-FL from the PostTCapoRF3 complex by mixing a large excess of unlabeled RF3 and GDPNP with a stoppedflow instrument and monitoring the increase in the fluorescence intensity of fluorescein (Figure 5B, blue trace). We observed a biphasic increase in the fluorescence intensity of fluorescein, which was analyzed by fitting a double exponential equation resulting in best-fit values for kobs1 and kobs2 (kobs1 = 2.7 ± 0.5 s-1 and kobs2 = 0.7 ± 0.3 s-1) (Table S1). As a control, we analyzed in parallel the dissociation kinetics of pre-bound RF3FLGDPNP from the PostTC (Figure 5B, violet trace). This gave us values for kobs1 and kobs2 that were identical to the values obtained for the dissociation of apo-RF3 from the PostTCapo-RF3 complex after mixing with GDPNP, described above. Because kobs1 and kobs2 were independent of whether GDPNP was pre-bound to RF3 or added later to apo-RF3 bound to the ribosome, it shows that the binding of GDPNP to apo-RF3 is not rate limiting for the dissociation of RF3 from the TC. Our result is consistent with a previous study that showed that nucleotide binding to apo-RF3 is fast 12. Importantly, the koff for RF1 was the same when the 70SRF1apo-RF3 complex was mixed with GTP or when the 70SRF1 complex was mixed with RF3GTP (Figure S3B). Thus, apo-RF3 bound to the termination complex is a good model to study the dissociation kinetics of RF3 with different guanine nucleotides. To confirm that the biphasic increase in the fluorescence intensity of fluorescein is caused by the decrease in FRET efficiency to rhodamine when RF3 dissociates from the PostTC, we performed dissociation experiment with RF3-FL bound to TC having


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unlabeled mRNA. As expected, the overall amplitude of the fluorescence signal was reduced by 7-fold with the unlabeled mRNA complex demonstrating that most of the signal with mRNA-RH and RF3-FL comes from FRET and is not due to changes in the microenvironment of fluorescein (Figure 5C, Figure S3C and S3D). We next analyzed the dissociation kinetics of apo-RF3 without any nucleotide or in the presence of GDP, GTP, GDPNP, or GTP-γ-S. Dissociation of apo-RF3 without nucleotide or with GDP showed a fast phase with >90% of the total amplitude and a slow phase with 10-fold by apo-RF3 compared to the absence of RF3. This result is consistent with the strong interaction observed between RF1 and apo-RF3 in the cryo-EM structure

26

. We also

show that apo-RF3 can bind GDP or GTP, which triggers the rapid dissociation of RF1 from the termination complex. Current models propose that the binding of GTP to apo-RF3 on the ribosome (or the direct binding of RF3GTP to the ribosome) triggers the dissociation of RF1 first, followed by GTP hydrolysis on RF3 and the dissociation of RF3GDP

9, 11, 12, 26, 33

.

However, the rate of RF3 dissociation was not determined in any of these studies, so it was unclear whether RF1 or RF3 dissociates first. We determined directly the rate


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constants for RF3 dissociation from the PostTC. Our data indicate that RF3 dissociation occurs in two steps: first the PostTCRF3 complex undergoes a conformation change followed by the second step, which is the dissociation of RF3 from the PostTC. Although we don’t know the details of the conformational change in the PostTCRF3 complex that we are observing in our kinetic studies, it is clear from structural data that both RF3 and the ribosome undergo large-scale conformational changes (described above). More importantly, the rate constant for RF3 dissociation is faster (kdiss = 1.7 s-1 with GTP) than the rate constant of RF1 dissociation (koff = 0.38 ± 0.04 s-1 with RF3GTP) indicating that RF3 dissociates first followed by RF1. Since the dissociation rate constant of RF3 is only 4-fold faster than the dissociation rate constant of RF1, it is likely that they are coupled events. Further insights into the sequence of events that occur during RF3-dependent release of RF1 from the ribosome come from structural studies. Structural data indicate that ribosome ratcheting occurs before GTP hydrolysis on RF3. Domain I of apo-RF3 bound to the unratcheted ribosomeRF1 complex does not contact the sarcin-ricin loop (SRL)

26

, whereas in the 70SRF3GDP(C/N)P structures

24, 25

, the ribosome is in the

ratcheted state and a direct contact is observed between domain I of RF3 and the SRL. Since the interaction of domain I with the SRL is important for GTP hydrolysis

21

, it

suggests that RF3GTP first induces ratcheting of the ribosome before GTP hydrolysis. Structural data taken together with our kinetic data indicate that the steps that lead to the release of RF1/RF2 from the ribosome are: (1) binding of RF3GTP to the ribosome, (2) ratcheting of the ribosome, (3) GTP hydrolysis on RF3, (4) dissociation of RF3GDP, and (5) dissociation of RF1/RF2 (Figure 6).


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It is also clear from structural data that ratcheting of the ribosome induced by RF3 destabilizes RF1/RF2 on the ribosome

10, 25

. Our kinetic data show that RF3 dissociates

before RF1 from the ribosome indicating that the ratcheted state is somehow maintained to promote the release of RF1. experiments

34

Structural data

10, 25

and single molecule FRET

have shown that ribosome ratcheting induced by RF3 causes the

deacylated tRNA, which is in the classical P/P state, to move to the hybrid P/E state. The hybrid P/E tRNA may stabilize the ribosome in the ratcheted state even after the dissociation of RF3. After the dissociation of RF3GDP, RF1/RF2 dissociates because of their weakened interactions with the ratcheted ribosome. Following the dissociation of RF1/RF2, the ratcheted ribosome with a hybrid P/E tRNA is the natural substrate for ribosome recycling factor (RRF) and elongation factor G (EF-G) in the final phase of protein synthesis, ribosome recycling.


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Acknowledgements

We thank Jack Kyte and Joseph Adams for discussions, and Venki Ramakrishnan, Rachel Green, Kristin Koutmou, Uli Muller and Seth Alexander for comments on the manuscript.

Funding Information This work was supported by National Science Foundation grant 1158127 to SJ.

Supporting Information Available Text, Table, Figures and Figure Legends


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References

[1] Youngman, E. M., McDonald, M. E., and Green, R. (2008) Peptide release on the ribosome: mechanism and implications for translational control, Annu Rev Microbiol 62, 353-373. [2] Petry, S., Weixlbaumer, A., and Ramakrishnan, V. (2008) The termination of translation, Curr Opin Struct Biol 18, 70-77. [3] Scolnick, E., Tompkins, R., Caskey, T., and Nirenberg, M. (1968) Release factors differing in specificity for terminator codons, Proc Natl Acad Sci U S A 61, 768774. [4] Rawat, U. B., Zavialov, A. V., Sengupta, J., Valle, M., Grassucci, R. A., Linde, J., Vestergaard, B., Ehrenberg, M., and Frank, J. (2003) A cryo-electron microscopic study of ribosome-bound termination factor RF2, Nature 421, 87-90. [5] Klaholz, B. P., Pape, T., Zavialov, A. V., Myasnikov, A. G., Orlova, E. V., Vestergaard, B., Ehrenberg, M., and van Heel, M. (2003) Structure of the Escherichia coli ribosomal termination complex with release factor 2, Nature 421, 90-94. [6] Laurberg, M., Asahara, H., Korostelev, A., Zhu, J., Trakhanov, S., and Noller, H. F. (2008) Structural basis for translation termination on the 70S ribosome, Nature 454, 852-857. [7] Weixlbaumer, A., Jin, H., Neubauer, C., Voorhees, R. M., Petry, S., Kelley, A. C., and Ramakrishnan, V. (2008) Insights into translational termination from the structure of RF2 bound to the ribosome, Science 322, 953-956. [8] Freistroffer, D. V., Pavlov, M. Y., MacDougall, J., Buckingham, R. H., and Ehrenberg, M. (1997) Release factor RF3 in E.coli accelerates the dissociation of release factors RF1 and RF2 from the ribosome in a GTP-dependent manner, Embo J 16, 4126-4133. [9] Zavialov, A. V., Buckingham, R. H., and Ehrenberg, M. (2001) A posttermination ribosomal complex is the guanine nucleotide exchange factor for peptide release factor RF3, Cell 107, 115-124. [10] Gao, H., Zhou, Z., Rawat, U., Huang, C., Bouakaz, L., Wang, C., Cheng, Z., Liu, Y., Zavialov, A., Gursky, R., Sanyal, S., Ehrenberg, M., Frank, J., and Song, H. (2007) RF3 induces ribosomal conformational changes responsible for dissociation of class I release factors, Cell 129, 929-941. [11] Koutmou, K. S., McDonald, M. E., Brunelle, J. L., and Green, R. (2014) RF3:GTP promotes rapid dissociation of the class 1 termination factor, RNA 20, 609-620. [12] Peske, F., Kuhlenkoetter, S., Rodnina, M. V., and Wintermeyer, W. (2014) Timing of GTP binding and hydrolysis by translation termination factor RF3, Nucleic Acids Res 42, 1812-1820. [13] Wilson, K. S., Ito, K., Noller, H. F., and Nakamura, Y. (2000) Functional sites of interaction between release factor RF1 and the ribosome, Nat Struct Biol 7, 866870. [14] Hetrick, B., Lee, K., and Joseph, S. (2009) Kinetics of stop codon recognition by release factor 1, Biochemistry 48, 11178-11184.


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[15] Eschenfeldt, W. H., Stols, L., Millard, C. S., Joachimiak, A., and Donnelly, M. I. (2009) A Family of LIC Vectors for High-Throughput Cloning and Purification of Proteins, Methods in molecular biology (Clifton, N.J.) 498, 105-115. [16] Powers, T., and Noller, H. F. (1991) A functional pseudoknot in 16S ribosomal RNA, EMBO J 10, 2203-2214. [17] Studer, S. M., and Joseph, S. (2007) Binding of mRNA to the bacterial translation initiation complex, Methods Enzymol 430, 31-44. [18] Studer, S. M., Feinberg, J. S., and Joseph, S. (2003) Rapid Kinetic Analysis of EFG-dependent mRNA Translocation in the Ribosome, J Mol Biol 327, 369-381. [19] Shi, X., Khade, P. K., Sanbonmatsu, K. Y., and Joseph, S. (2012) Functional role of the sarcin-ricin loop of the 23S rRNA in the elongation cycle of protein synthesis, J Mol Biol 419, 125-138. [20] Kuzmic, P. (1996) Program DYNAFIT for the analysis of enzyme kinetic data: application to HIV proteinase, Anal Biochem 237, 260-273. [21] Voorhees, R. M., Schmeing, T. M., Kelley, A. C., and Ramakrishnan, V. (2010) The mechanism for activation of GTP hydrolysis on the ribosome, Science 330, 835838. [22] Voorhees, R. M., Schmeing, T. M., Kelley, A. C., and Ramakrishnan, V. (2011) Response to Comment on "The Mechanism for Activation of GTP Hydrolysis on the Ribosome", Science 333, 37. [23] Liljas, A., Ehrenberg, M., and Aqvist, J. (2011) Comment on "The Mechanism for Activation of GTP Hydrolysis on the Ribosome", Science 333, 37. [24] Zhou, J., Lancaster, L., Trakhanov, S., and Noller, H. F. (2012) Crystal structure of release factor RF3 trapped in the GTP state on a rotated conformation of the ribosome, Rna 18, 230-240. [25] Jin, H., Kelley, A. C., and Ramakrishnan, V. (2011) Crystal structure of the hybrid state of ribosome in complex with the guanosine triphosphatase release factor 3, Proc Natl Acad Sci U S A 108, 15798-15803. [26] Pallesen, J., Hashem, Y., Korkmaz, G., Koripella, R. K., Huang, C., Ehrenberg, M., Sanyal, S., and Frank, J. (2013) Cryo-EM visualization of the ribosome in termination complex with apo-RF3 and RF1, eLife 2, e00411. [27] Leipe, D. D., Wolf, Y. I., Koonin, E. V., and Aravind, L. (2002) Classification and evolution of P-loop GTPases and related ATPases, J Mol Biol 317, 41-72. [28] Kisselev, L. L., and Buckingham, R. H. (2000) Translational termination comes of age, Trends Biochem Sci 25, 561-566. [29] Mikuni, O., Ito, K., Moffat, J., Matsumura, K., McCaughan, K., Nobukuni, T., Tate, W., and Nakamura, Y. (1994) Identification of the prfC gene, which encodes peptide-chain-release factor 3 of Escherichia coli, Proc Natl Acad Sci U S A 91, 5798-5802. [30] Grentzmann, G., Brechemier-Baey, D., Heurgue, V., Mora, L., and Buckingham, R. H. (1994) Localization and characterization of the gene encoding release factor RF3 in Escherichia coli, Proc Natl Acad Sci U S A 91, 5848-5852. [31] Zaher, H. S., and Green, R. (2011) A primary role for release factor 3 in quality control during translation elongation in Escherichia coli, Cell 147, 396-408. [32] Zaher, H. S., and Green, R. (2009) Quality control by the ribosome following peptide bond formation, Nature 457, 161-166. 31 ACS Paragon Plus Environment

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[33] Gao, N., Zavialov, A. V., Ehrenberg, M., and Frank, J. (2007) Specific interaction between EF-G and RRF and its implication for GTP-dependent ribosome splitting into subunits, J Mol Biol 374, 1345-1358. [34] Sternberg, S. H., Fei, J., Prywes, N., McGrath, K. A., and Gonzalez, R. L., Jr. (2009) Translation factors direct intrinsic ribosome dynamics during translation termination and ribosome recycling, Nat Struct Mol Biol 16, 861-868.


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Three independent experiments were performed to generate the mean ± SD. Each independent experiment is the average of four replicas of rapid mixing.


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Three independent experiments were performed for GTP and GDPNP, and two independent experiments were performed for GTPγ-S, GDP and no nucleotides to generate the mean ± SD. Each independent experiment is the average of four replicas of rapid mixing. The rate constants were determined by fitting to Scheme 1 using numerical analysis.


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Figure Legends

Figure 1. Binding of GTP to RF3. (A)-(D) Emission spectrum showing the changes in fluorescence intensity because of mantGTP binding to wild type RF3, RF3 (His92Ala), RF3 (Ser69Ala) and RF3 (Thr27Ala) mutants. Black trace, control with only 250 nM mantGTP solution; green trace, 250 nM mantGTP mixed with 500 nM RF3; red trace, 250 nM mantGTP, 500 nM RF3 and 5 µM GTP used as competition for binding to RF3 (chase). The x-axis shows the wavelength in nm and the y-axis shows the fluorescence intensity in arbitrary units (a.u.). (E) Control experiment with RF1, which should not bind RF3. Black trace, control with only 250 nM mantGTP solution; green trace, 250 nM mantGTP mixed with 500 nM RF1; red trace, 250 nM mantGTP, 500 nM RF1 and 5 µM GTP. (F) Bar graph showing the % change in fluorescence intensity when mantGTP binds to RF3. The green and red bars represent mantGTP bound to wild type and the indicated RF3 mutants in the absence of GTP and in the presence of GTP, respectively. The error bars show mean ± SD from two independent experiments.

Figure 2.

Binding of RF3 to ribosomes.

(A) Binding of wild type RF3, RF3

(His92Ala), RF3 (Ser69Ala) and RF3 (Thr27Ala) mutants to PostTC. SDS-PAGE gels showing the binding of RF3 to ribosomes with GDP, GDPCP, or GTP. (-) and (+) indicate the absence and presence of RF1 in the binding reaction, respectively. Marker lane shows the molecular weight ladder. Positions of RF1, RF3, ribosomal protein S1 and S2 are indicated on the right of the gel. (B) Bar graph showing the extent of RF3 bound to ribosomes with the different guanine nucleotides in the absence (-) or presence (+) of RF1.

Wild type RF3 (black bar), RF3 (His92Ala) mutant (green bar), RF3 35 ACS Paragon Plus Environment

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(Ser69Ala) mutant (magenta bar) and RF3 (Thr27Ala) mutant (orange bar). The y-axis shows fraction of RF3 bound relative to ribosomal protein S2, and the error bars show mean ± SD from two independent experiments.

Figure 3. Kinetics of GTP hydrolysis by ribosomeRF3 complex. (A) Representative time courses of GTP hydrolysis by wild type RF3, RF3 (His92Ala), RF3 (Ser69Ala) and RF3 (Thr27Ala) mutants in the presence of PostTC. The concentration of wild type and mutant RF3 is 20 µM in these time courses. The x-axis shows time in minutes and the yaxis shows GTP hydrolyzed in picomoles. (B) Graph showing the kinetics of GTP hydrolysis by the single cysteine RF3 with PostTC having tRNAfMet (square) or tRNAPhe (circle) in the P site. The x-axis shows RF3 concentration and the y-axis shows the initial velocity of GTP hydrolysis derived from the time courses. The error bars show mean ± SD from three independent experiments.

Figure 4. FRET assay for monitoring RF1 dissociation from the ribosome. (A) Emission spectrum showing the changes in fluorescence intensity because of RF1 binding to the ribosome.

Control reaction with unlabeled ribosome complex and

unlabeled RF1 (black trace); control reaction with unlabeled ribosome complex and fluorescein labeled RF1 (green trace); control reaction with rhodamine labeled ribosome complex and unlabeled RF1 (blue trace); reaction with rhodamine labeled ribosome complex and fluorescein labeled RF1 (red trace). The x-axis shows the wavelength in nm and the y-axis shows the fluorescence intensity in counts per second (cps). (B) Time course of RF3-promoted dissociation of RF1 from the ribosome. The increase in the


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emission intensity of fluorescein attached to RF1 was monitored.

Control reaction

without RF3 (black trace); RF3GDP (orange trace); RF3GDPNP (blue trace); RF3 (His92Ala) mutant with GTP (green trace); RF3GTP (red trace). In all cases, black lines show the fit to a single exponential equation. (C) Apo-RF3 decreases the rate of RF1 dissociation from the ribosome. Time course of RF1 dissociation in the absence RF3 (black trace); time course of RF1 dissociation in the presence of RF3GDP (red trace); and time course of RF1 dissociation in the presence of apo-RF3 (green trace). (D) Apo-RF3 bound to the ribosome can bind GDP or GTP to accelerate the dissociation of RF1. Red trace shows the dissociation kinetics of fluorescein labeled RF1 bound to ribosome up the addition of unlabeled RF1, RF3 and GDP at the indicated time (arrow, +RF3GDP); green trace shows that the dissociation rate of fluorescein labeled RF1 decreases upon addition of unlabeled RF1 and apo-RF3 (arrow, + apo-RF3), and then increases upon the addition of GTP (arrow, +GTP).

Figure 5. FRET assay for monitoring RF3 dissociation from the ribosome. (A) Emission spectrum showing the changes in fluorescence intensity because of RF3 binding to the ribosome.

Control reaction with unlabeled termination complex and

unlabeled RF3 (black trace); control reaction with unlabeled termination complex and fluorescein labeled RF3 (RF3-FL) (green trace); control reaction with rhodamine labeled termination complex (TC-RH) and unlabeled RF3 (blue trace); and reaction with rhodamine labeled termination complex and fluorescein labeled RF3 (red trace). The xaxis shows the wavelength in nm and the y-axis shows the fluorescence intensity in counts per second (cps).

(B) Time course of RF3 dissociation from ribosome.


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Dissociation of fluorescein labeled apo-RF3 from ribosome after the addition of unlabeled RF3 and GDPNP (blue trace); dissociation of fluorescein labeled RF3GDPNP after the addition of unlabeled RF3 and GDPNP (violet trace). (C) Control experiment showing that the increase in fluorescein fluorescence intensity is due to the dissociation of RF3 from the ribosome. Time course of fluorescein labeled RF3 dissociation from rhodamine labeled ribosome complex (mRNA-RH) upon the addition of unlabeled RF3 and GDPNP (blue trace); time course of fluorescein labeled RF3 dissociation from unlabeled ribosome complex (mRNA) upon the addition of unlabeled RF3 and GDPNP (light blue trace). nucleotides.

(D) Time course of apo-RF3 dissociation with different guanine

Dissociation of fluorescein labeled apo-RF3 from ribosome after the

addition of unlabeled RF3 without any guanine nucleotide (black trace); dissociation of fluorescein labeled apo-RF3 from ribosome after the addition of unlabeled RF3 and GDPNP (blue trace); dissociation of fluorescein labeled apo-RF3 from ribosome after the addition of unlabeled RF3 and GTP (red trace); dissociation of fluorescein labeled apoRF3 from ribosome after the addition of unlabeled RF3 and GDP (orange trace); dissociation of fluorescein labeled apo-RF3 from ribosome after the addition of unlabeled RF3 and GTP-γ-S (grey trace).

In all cases, black lines show the fit to a double

exponential equation.

Figure 6. Mechanism of RF3-promoted dissociation of RF1/RF2 from the ribosome. Translation termination begins with RF1 or RF2 binding to ribosome with a stop codon in the A site and the release of the nascent peptide attached to the tRNA in the P site. RF3GTP then binds to the post-termination complex (bottom scheme). Alternatively,


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RF3GTP binds to the ribosome before peptide release followed by peptide release by RF1/RF2 (top scheme). RF3GTP then induces ratcheting of the 30S subunit relative to the 50S subunit, which stabilizes the deacylated tRNA in P/E hybrid state. Next, RF3 hydrolyzes GTP and dissociates from the ribosome.

Finally, RF1/RF2 dissociates

leaving behind a ratcheted ribosome with a hybrid P/E tRNA.

Color scheme: 50S

ribosomal subunit (grey), unratcheted 30S ribosomal subunit (cyan), ratcheted 30S ribosomal subunit (violet), tRNA (green), nascent peptide (black circles), mRNA (blue line), RF1/RF2 (red), and RF3 (yellow). The three tRNA binding sites (E, P, and A) are also indicated.


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Figure 1 180x255mm (300 x 300 DPI)

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Figure 2 196x239mm (300 x 300 DPI)


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Figure 3 163x118mm (300 x 300 DPI)


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Figure 4 172x203mm (300 x 300 DPI)


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Figure 5 175x194mm (300 x 300 DPI)


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Figure 6 234x88mm (300 x 300 DPI)


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For Table of Contents Use Only

RF1 dissociation

RF1 RF3 GDP dissociation 50S

E P A 30S

E P A

E P A

RF3 GTP

Mechanism of Translation Termination: RF1 Dissociation follows RF3 Dissociation from the Ribosome Xinying Shi and Simpson Joseph


Mechanism of Translation Termination: RF1 Dissociation Follows

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