Multiple Parallel Pathways of Translation Initiation on the CrPV IRES Alexey Petrov, Rosslyn Grosely, Jin Chen, Seán E. O’Leary, Joseph D. Puglisi  Molecular Cell  Volume 62, Issue 1, Pages 92-103 (April 2016) DOI: 10.1016/j.molcel.2016.03.020 Copyright © 2016 Elsevier Inc. Terms and Conditions

Molecular Cell 2016 62, 92-103DOI: (10.1016/j.molcel.2016.03.020) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 1 Subunit Recruitment and 80S Formation (A) Experimental schematics and example trace of 40S ribosomal subunit binding to the CrPV IRES. CrPV IRES-Cy5-biotin (red) was immobilized on the surface of a ZMW slide, and 40S-Cy3 (green) subunits were delivered to it. The colors in the schematics match the dye pseudocolors in the example trace. The text below the x axis denotes complex evolution over time. In the example trace, 40S binding is identified as a burst of green fluorescence at ∼40 s. (B) 40S:CrPV IRES complexes were immobilized on the surface. In this and all subsequent experiments, the complexes were immobilized via 3′-biotinylated CrPV IRES. 60S-Cy5 (red) subunits were delivered to 40S-Cy3:CrPV IRES-biotin complexes (green) immobilized on the surface of a ZMW chip. 60S joining is identified as a burst of red fluorescence at ∼60 s in the example trace. (C) The 40S subunit arrival time distribution (blue dots) was fit to a single exponential model (red line). n = 142, where n is the number of molecules used to build the distribution. (D) The 60S subunit arrival time distribution (blue dots) was fit to a double exponential model (red line). n = 302. (E) The kobs for 60S subunit arrival at 200 nM and 20 nM (left bar plot) was determined by fitting the data to a double exponential model. Error bars are 95% confidence interval of the fit. The effect of subunit concentration on joining efficiency is shown in the bar plot on the right. 60S joining efficiency is the ratio of 80S:IRES complexes to 40S:IRES complexes. Molecular Cell 2016 62, 92-103DOI: (10.1016/j.molcel.2016.03.020) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 2 80S:CrPV IRES Assembly Pathways 40S-Cy3.5 (yellow) and 60S-647 (red) subunits were codelivered to CrPV IRES-Cy3-biotin (green) immobilized on the surface of a ZMW chip. (A) Example trace showing sequential arrival of the 40S subunit (burst of yellow fluorescence at ∼90 s) followed by arrival of the 60S subunit (burst of red fluorescence at 105 s) to the CrPV IRES. (B) In the example trace, the 80S ribosome is directly recruited to the CrPV IRES. Coarrival of the 40S and 60S ribosomes to the CrPV IRES is indicated by the simultaneous burst of yellow and red fluorescence at ∼40 s. Red fluorescence photobleaching occurs at ∼300 s, whereas green and yellow fluorescence persist for the length of the movie. A yellow fluorescence blink occurs at ∼160 s. (C) The Mg2+ dependence of simultaneous subunit recruitment by the CrPV IRES is shown as the percentage of 80S arrivals that occurred through the coarrival of the 40S and 60S subunits. n = 68, 283, and 57, correspondingly. (D) Model of 40S (yellow) and 60S (blue) ribosome recruitment by the CrPV IRES (red). Arrow width indicates pathway flux. Molecular Cell 2016 62, 92-103DOI: (10.1016/j.molcel.2016.03.020) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 3 Following Initiation and Elongation in Real Time 40S-Cy3.5 (yellow) and 60S-Cy5 (red) ribosomes and ternary complex (tRNAPhe-Phe-Cy3 [green], eEF1A, and eEF1Bα) were codelivered to CrPV IRES-Cy5.5 (magenta)-biotin immobilized on the surface of a ZMW chip. (A) Binding of tRNA (burst of green fluorescence at 185 s) following the sequential arrival of the 40S (burst of yellow fluorescence at 45 s) and 60S (burst of red fluorescence at 120 s) ribosomal subunits to the CrPV IRES is shown in the example trace. (B) In the example trace, simultaneous arrival of the 40S and 60S subunits (burst of red and yellow fluorescence at 110 s) to the immobilized IRES is rapidly followed by tRNA binding to the 80S-CrPV IRES complex (burst of green fluorescence at 130 s). (C) tRNA recruitment efficiencies; n = 673. Molecular Cell 2016 62, 92-103DOI: (10.1016/j.molcel.2016.03.020) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 4 eEF2 Is Required for Efficient Elongator tRNA Binding by 80S-CrPV IRES Complexes (A) Testing eEF2 requirement; the experiment schematics. 40S:60S-Cy5:CrPV IRES-biotin complexes were pre-assembled in bulk. Complexes were surface immobilized and washed with reaction buffer. tRNAPhe-Phe-Cy3 ternary complex was delivered with or without eEF2. Binding efficiency was measured as the percentage of 80S:CrPV IRES complexes that bound tRNA. (B) tRNA binding efficiency of 80S:CrPV complexes. n = 200, 860, 233, 746, and 617. (C) Testing the role of eEF2 in stabilization of translocated state. 80S:CrPV IRES complexes were assembled in the presence or absence of eEF2 and then surface immobilized. The unbound complexes were washed off. tRNAPhe-Phe-Cy3 ternary complex was delivered with or without eEF2. (D) tRNA binding efficiencies in 0 and +1 frames; n = 179, 573, 388, 567, and 1,200. The legend below the x axes denotes eEF2 presence during the reaction. eEF1A and GTP were ubiquitously present unless otherwise noted above the corresponding column. Molecular Cell 2016 62, 92-103DOI: (10.1016/j.molcel.2016.03.020) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 5 tRNA Fluctuations (A) In total, 98% of all tRNA traces began in a high-intensity state. Measured state lifetimes were used to calculate the percentage of traces in which the first state is expected to be shorter than exposure time (0.1 s), thus being unobservable. This equals 1.4% of the traces. (B) tRNA state dwell-time dependence on state position. The tRNA trace schematic is shown in green. Bars depict dwell times. Error bars are 95% confidence interval of the fit. (C) Combined low-intensity state dwell times (n = 1,155) fit a single exponential model indicating a single-step process. (D) Combined high-intensity dwell times (n = 1,136) are best described by a double exponential model, shown in blue. This indicates either multiple subpopulations in the high-intensity state and/or a multistep process for high-to-low state transition. The single exponential model is shown in red for comparison. The first long state was excluded from the fit. A single exponential model was used to calculate the individual state lifetimes shown in (B) due to the poor robustness of the double exponential fit for the individual states. Molecular Cell 2016 62, 92-103DOI: (10.1016/j.molcel.2016.03.020) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 6 Following Frame Selection in Real Time 60S-Cy3 (green) ribosomes, elongation factors, and either 0 frame tRNA (tRNAVal-Cy5 [red]) or +1 frame tRNA (tRNAPhe-Cy3.5 [yellow]) was delivered to 40S-Cy5.5:CrPV IRES-biotin complexes (magenta) immobilized on the surface of a ZMW slide. (A) Example trace of 0 frame tRNAVal-Cy5 (red) delivery. (B) Arrival efficiency of 0 frame tRNA at various eEF2 concentrations. n = 772, 778, 874, and 776. (C) Arrival rate of 0 frame tRNA at various eEF2 concentrations. n = 229, 287, and 198. (D) Example trace of +1 frame tRNAPhe-Cy3.5 (yellow) delivery. (E) Arrival efficiency of +1 frame tRNA at various eEF2 concentrations. n = 738, 673, 595, and 648. (F) Arrival rate of +1 frame tRNA at various eEF2 concentrations. n = 105, 123, and 103. (G) Zero frame (tRNAVal-Cy5 [red]) and +1 frame tRNA (tRNAPhe-Cy3.5 [yellow]) were codelivered along with 60S-Cy3 (green) subunits and elongation factors to 40S-Cy5.5:CrPV IRES-biotin complexes (magenta) immobilized on the surface of a ZMW slide to follow frame selection in real time. The pathway selection, as it was observed, is represented by the flow chart; n = 312. Error bars are 95% confidence interval of the fit. Molecular Cell 2016 62, 92-103DOI: (10.1016/j.molcel.2016.03.020) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 7 Multiple Pathways of Translation Initiation by the CrPV IRES 80S assembly on the CrPV IRES occurs via sequential recruitment of the 40S and 60S ribosomal subunits, and by direct recruitment of the 80S ribosome. Regardless of the assembly pathway, translocation is required for tRNA acceptance by 80S:IRES complexes. The translocation step can occur spontaneously in an eEF2-independent manner. The translocated complexes are unstable, undergo back-translocation, and lack a defined reading frame. The frame ambiguity during the first translocation results in 80S:CrPV IRES complexes that can accept 0 or +1 frame tRNA. The incoming tRNA captures and stabilizes the translocated state of the ribosome. The reading frame is transiently set by the step preceding tRNA binding, and frame-selection efficiency depends on the relative rates of 0 and +1 frame tRNA arrival to the A site. Molecular Cell 2016 62, 92-103DOI: (10.1016/j.molcel.2016.03.020) Copyright © 2016 Elsevier Inc. Terms and Conditions