1 2 3 4 5 6 7 8 Supplementary Materials for Rapid 40S scanning and its regulation by mRNA structure during eukaryotic translation initiation Jinfan Wang et al. 9 Corresponding author: Joseph D. Puglisi ([email protected]) 10 11 12 13 14 15 16 17 18 19 20 21 This file includes: Materials and Methods Figs. S1 to S13 Table S1 References (47-57) 1 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 Materials and Methods 1. Yeast translation factors Yeast Saccharomyces cerevisiae eIFs 1, 1A, 2, 3, 4A, 4B, 4E, 4G, 5 and 5B, and eEFs 1A and 1Bα were prepared according to previously established methods (21). To express Pab1p or Hcr1p, the plasmid pTYB2-Pab1p (Addgene #37234) or pTYB2-HCR1 (Addgene #37233) were transformed into Escherichia coli Rosetta2 (DE3) cells (Novagen), and the proteins were purified as previously described (47, 48). Full-length DED1 gene was PCR-amplified from yeast genomic DNA and cloned into a pET28c plasmid for the expression of a recombinant 6His-MBP-TEV protease site-Ded1p fusion protein. The resulting plasmid was transformed into Rosetta2 (DE3) cells and overexpressed by induction at an OD600nm = 0.5 with 1 mM IPTG at 16ºC for 16 hours. Cells were pelleted by centrifugation at 5000  g for 12 min, resuspended in 30 mL of lysis buffer (50 mM HEPES-KOH pH 7.5, 300 mM NaCl, 10 mM imidazole, 5 mM 2-mercaptoethanol) supplemented with a cOmplete EDTA free protease inhibitor cocktail tablet (Roche) and lysed by sonication (MISONIX, 2 s on / 6 s off, 100 s total on time at 80% amplitude). The lysate was clarified by centrifugation at 41,656  g for 30 min at 4 °C in a F21-850y rotor (Thermo Fisher Scientific). The clarified lysate was loaded to a 2.5 mL Ni-NTA (Qiagen) gravity flow column equilibrated with lysis buffer. The column was washed with 50 mL wash buffer 1 (50 mM HEPES-KOH pH 7.5, 1000 mM NaCl, 20 mM imidazole, 5 mM 2-mercaptoethanol) and 20 mL wash buffer 2 (50 mM HEPES-KOH pH 7.5, 300 mM NaCl, 5 mM 2-mercaptoethanol). Bound proteins were eluted with a buffer containing 50 mM HEPES-KOH pH 7.5, 300 mM NaCl, 200 mM imidazole and 5 mM 2-mercaptoethanol. The eluate was diluted with 4 volumes of buffer Q-A (50 mM Tris-HCl pH 8.0, 5 mM 2-mercaptoethanol) and loaded to a 5 mL HiTrap Q HP column equilibrated with buffer Q-AB (50 mM Tris-HCl pH 8.0, 100 mM NaCl, 5 mM 2mercaptoethanol). The protein was eluted by applying an 80 mL linear gradient from 100% buffer Q-AB to 100% buffer Q-B (50 mM Tris-HCl pH 8.0, 500 mM NaCl, 5 mM 2mercaptoethanol). The 6His-MBP tag was cleaved with TEV protease and removed by flowing through the samples on a Ni-NTA column. The purified Ded1p was dialyzed twice against the storage buffer (10 mM HEPES-KOH pH 7.3, 200 mM KOAc, 2 mM DTT, 50% glycerol) and stored at -80ºC after liquid nitrogen freezing. The ATPase activity of the protein was verified by a malachite green assay as described (49). 2. Yeast ribosomal subunits We have previously labeled and characterized the ybbR-tagged yeast 40S subunit (at the Nterminus of uS19) and 60S subunit (at the C-terminus of uL18) (21). Here, for double tagging and labeling of the 40S subunit, the S6 tag (amino acid sequence of GDSLSWLLRLLN) was genetically fused to the N-terminus of uS19 using the same strategy as described for the ybbR tagging of uS19. The resulting yeast strain was transformed with a dsDNA carrying a URA3 cassette to tag the C-terminus of uS5 with the A1 tag (amino acid sequence of GDSLDMLEWSLM) via homologous recombination using standard PCR-based methods (50), and selected on SC-His-URA plates. Ribosome subunits were purified as previously described (21). A two-step labeling protocol was developed to obtain the Cy3.5-S6-uS19 and uS5-A1-Cy5 doubly labeled 40S subunits (51). First, 1 µM of the purified double-tagged 40S subunit was mixed with 2 µM SFP synthase and 10 µM Cy3.5-CoA in a reaction buffer containing 50 mM HEPES-KOH pH 7.5, 10 mM MgCl2 and 1 mM DTT. The reaction was incubated at 30ºC for 10 min before the sample was loaded on top of a sucrose cushion (containing 30 mM HEPES-KOH 2 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 pH 7.5, 100 mM KOAc, 5 mM Mg(OAc) 2, 2 mM DTT and 0.5 M sucrose). The ribosomes were pelleted by ultracentrifugation at 351,955  g and 4 °C for 60 min in a TLA100.2 rotor, and the res