Volume 20, Issue 1, Pages 161-172 (July 2017) Post-termination Ribosome Intermediate Acts as the Gateway to Ribosome Recycling  Arjun Prabhakar, Mark C. Capece, Alexey Petrov, Junhong Choi, Joseph D. Puglisi  Cell Reports  Volume 20, Issue 1, Pages 161-172 (July 2017) DOI: 10.1016/j.celrep.2017.06.028 Copyright © 2017 The Authors Terms and Conditions

Cell Reports 2017 20, 161-172DOI: (10.1016/j.celrep.2017.06.028) Copyright © 2017 The Authors Terms and Conditions

Figure 1 Experimental Setup (A) In all experiments, 30S preinitiation complexes (PIC) containing Cy3B-30S, fMet-tRNAfMet, and IF2-GTP is immobilized on the surface of the ZMW wells through biotinylated mRNAs. The reaction is started by delivery of BHQ-2-50S, Phe-TC, EF-G, and Cy5-RF1 or Cy5-RF2. (B) Structure of RF2-bound 70S ribosome (Korostelev et al., 2008) showing the C-terminal fluorescent labeling position (C367). (C) Bottom: representative trace of Cy3B- and BHQ-2-labeled ribosome translating in the presence of Phe-TC, EF-G, and Cy5-RF. Top: schematic of translation, with ribosome states linked to fluorescence signals in the trace. Delivery of reagents results in initiation through 50S subunit joining (shown by quenching of green Cy3B signal), followed by one round of elongation (reported by a cycle of low and high Cy3B intensities), then binding of Cy5-RF during termination (shown by red Cy5 signal, see also Figure S1). Departure of Cy5-RF after peptide release correlates with ribosome rotation, presenting a post-termination rotated-state intermediate (highlighted in gray). Cy5-RF can rebind to this intermediate to induce ribosome rotation back to non-rotated state. RRF and EF-G bind to this post-termination rotated ribosome to catalyze 50S subunit dissociation. The boxed area shows the mRNA sequences used in these experiments. Cell Reports 2017 20, 161-172DOI: (10.1016/j.celrep.2017.06.028) Copyright © 2017 The Authors Terms and Conditions

Figure 2 RF Rebinding Inhibits 70S Ribosome Disassembly after Termination (A) Schematic describing the two competing pathways from the post-termination rotated-state intermediate in the absence of any recycling factors. The observed rate of disappearance (kobs) of this intermediate is equal to the sum of the rates of the two competing pathways: RF rebinding (kpostRF,on[RF]) and spontaneous 50S subunit dissociation (k50S,off). (B) Representative trace highlighting the post-termination rotated states. (C) Post-synchronization of post-termination rotated state dwell times depict an exponential decay of post-termination rotated state population. This distribution was fit to a single-exponential equation to obtain kobs. (D) Plot of kobs as a function of [RF2]. The slope and y-intercept of the least-squares linear fit to the plot represent the RF-rebinding rate (kpostRF,on) and 50S subunit dissociation rate (k50S,off), respectively. Error bars represent SD. Cell Reports 2017 20, 161-172DOI: (10.1016/j.celrep.2017.06.028) Copyright © 2017 The Authors Terms and Conditions

Figure 3 RRF and EF-G Block RF Rebinding and Catalyze Subunit Disassembly (A) Percentage of ribosomes showing RF rebinding at 0.05 and 1 μM EF-G as a function of [RRF]. (B) Percentage of ribosomes showing RF rebinding at 0, 1, and 20 μM RRF as a function of [EF-G]. (C) Subunit disassembly time at 0.05, and 1 μM EF-G as a function of [RRF]. Subunit disassembly time is defined as the mean dwell time of the post-termination rotated state before 50S subunit dissociation. Error bars represent SEM. (D) Subunit disassembly time at 1 and 20 μM RRF plotted as a function of [EF-G]. Error bars represent SEM. Cell Reports 2017 20, 161-172DOI: (10.1016/j.celrep.2017.06.028) Copyright © 2017 The Authors Terms and Conditions

Figure 4 EF-G Binds to RRF-Bound Ribosome to Catalyze Subunit Disassembly by GTP Hydrolysis (A) Representative trace of 50 nM Cy5-EF-G-GTP experiment in the presence of 20 μM RRF (and 10 nM wild-type RF2, 50 nM Phe TC, and 100 nM BHQ-2) showing Cy5-EF-G pulses during elongation and recycling. During recycling, Cy5-EF-G binds to the post-termination rotated state and is correlated with 50S subunit dissociation (boxed area). (B) Post-synchronization of subunit disassembly signal and Cy5-EF-G(GTP) occupancy in the presence of RRF as boxed in (A). (C) Representative trace showing multiple rapid pulses of unproductive Cy5-EF-G(GTP) binding to the post-termination rotated state in the absence of RRF. (D) Representative trace showing long occupancy of Cy5-EF-G(GDPNP) to the rotated state both in presence and absence of RRF. FRET is observed between Cy5-EF-G and Cy3B-30S. (E) Mean dwell time of Cy5-EF-G pulses in the presence or absence of 20 μM RRF and different guanine nucleotides (G-ntd): GTP or GDPNP. Error bars represent SEM. Cell Reports 2017 20, 161-172DOI: (10.1016/j.celrep.2017.06.028) Copyright © 2017 The Authors Terms and Conditions

Figure 5 IF3 Binds to 30S-tRNA-mRNA Complex to Induce tRNA Departure, followed by 30S Subunit Dissociation (A) Representative trace showing that the Cy5.5-tRNAPhe does not depart in the presence of 1 μM RRF and 500 nM EF-G without IF3. Cy5.5-tRNAPhe presence is necessary for the observed reversibility of 50S subunit dissociation and Cy5-RF2 rebinding. See also Figure S7A. (B) Mean lifetime of Cy5.5 fluorescence decreases in the presence of 1 μM IF3. Error bars represent SEM. (C) Representative trace showing that BHQ-2-50S subunit dissociation is followed by sequential departures of the Cy5.5-tRNAPhe and Cy3B-30S subunit in the presence of 1 μM RRF, 500 nM EF-G, and 1 μM IF3. Inset: delay between 50S subunit dissociation and tRNA departure (blue line) and delay between tRNA departure and 30S subunit dissociation (orange line) are highlighted. See also Figure S7B. (D) Mean tRNA departure time, defined as the delay time between 50S subunit dissociation and tRNA departure (blue), is dependent on IF3 concentration. Mean 30S subunit departure time, defined as the delay time between tRNA departure and 30S subunit dissociation (orange), is independent of IF3 concentration. Error bars represent SEM. Cell Reports 2017 20, 161-172DOI: (10.1016/j.celrep.2017.06.028) Copyright © 2017 The Authors Terms and Conditions

Figure 6 The Complete Model of Ribosome Recycling Post-termination rotated-state 70S intermediate is the substrate for RRF and EF-G to bind and catalyze subunit disassembly. Although EF-G can bind to both RRF-free and RRF-bound 70S complex, RRF and EF-G must bind sequentially to catalyze subunit disassembly. After 50S subunit dissociation, IF3 binds to the remaining 30S complex to induce P-site tRNA dissociation, followed by spontaneous dissociation of 30S subunit from the mRNA. The numerical values of the kinetic parameters here are presented in Table 1. Cell Reports 2017 20, 161-172DOI: (10.1016/j.celrep.2017.06.028) Copyright © 2017 The Authors Terms and Conditions