doi: 10.1038/nature07298
SUPPLEMENTARY INFORMATION Concurrent Nucleation of 16S Folding and Induced Fit in 30 S Ribosome Assembly Tadepalli Adilakshmi,1,3 Deepti L. Bellur2 and Sarah A. Woodson1* T.C. Jenkins Department of Biophysics and 2Program in Cell, Molecular and Developmental Biology and Biophysics, Johns Hopkins University, 3400 N. Charles St., Baltimore, MD 218182685 USA 1
FULL METHODS Purification of total 30S proteins and 16S rRNA. 70S ribosomes were isolated from E. coli MRE 600 as previously described.1 Ribosomes (60-90 mg) were dissociated into subunits by dialyzing 4 h with two buffer changes against Buffer E (50 mM Tris-HCl, pH 7.5, 150 mM NH4Cl, 1 mM MgCl2, 6 mM β-mercaptoethanol), and the subunits were isolated from each other by centrifugation at 28,000 rpm 16 h on a 10-40% linear sucrose gradient in the same buffer. Fractions containing 30S subunits were pooled and MgCl2 concentration raised to 10 mM. Subunits were pelleted at 40,000 rpm for 16 h (Beckman Ti-60), and dissolved in 0.5 mL 20 mM Tris-HCl, pH 7.5, 10 mM NH4Cl, 10 mM MgCl2, 0.5 mM EDTA and 6 mM β-mercaptoethanol by slowly rocking on ice. Aliquots were frozen in dry ice/ethanol bath and stored at –80 ˚C. Total 30S protein and 16S rRNA were isolated from purified 30S particles as described.1 Analytical sucrose gradients. Sucrose stocks (10% and 40%) were prepared in Buffer E, and linear gradients prepared using a Gradient Master (BioComp Instruments, New Brunswick, CA). Native 30S ribosomal subunits or reconstituted subunits (100 pmol 16S rRNA plus 150 pmol TP30 in 400 µL) were layered on the top of the gradient, and centrifuged at 28,000 rpm for 16 h, 4 ˚C, in a SW-28 rotor (Beckman). Peptidyl transferase activity. Initiation complexes were formed by incubating native or reconstituted 30S (0.5 µM) with native 50S (0.5 µM) and initiation factors, a short mRNA, GTP and 0.5 µM [35S]-fMet-tRNAfMet at 37 ˚C for 45 min as previously described.2 The elongation ternary complex was prepared separately with 4.5 µM tRNA(phe), 5 µM phenylalanine, E. coli S100 extract and ATP regeneration system, 45 min at 37 ˚C.3 Equal volumes of initiation and elongation complexes were mixed and incubated at 20 ˚C for 5 s to 5 min. At various intervals, reaction aliquots were quenched with 0.5 N KOH, 37 ˚C, 20 min to hydrolyze the peptidyl tRNA. Dipeptide bond formation was monitored by electrophoresis of the hydrolyzed tRNA on TLC-cellulose with pyridine/acetic acid at 800 V, 1 h.2 Time-resolved hydroxyl radical footprinting. Time-resolved footprinting experiments were carried out as previously described4,5 at beamlines X28C at the National Synchrotron Light Source (Brookhaven National Laboratory, Upton, NY). 16S rRNA (50 pmol or 0.25 µM) was allowed to fold in 80 mM K-cacodylate, pH 7.5, 330 mM KCl, 20 mM MgCl2, 6 mM βmercaptoethanol, 0.01% Nikkol for 15 min at 42 ˚C. Assembly was initiated at 30 ˚C by rapid mixing of 20 µL TP30 (75 pmol or 0.38 µM each protein). Mixing was carried out using a water-jacketed rapid quench apparatus (Kin-Tek) modified to contain an X-ray flow cell downstream of the mixing tee. After a mixing delay of 10 ms to 180 s, the 40 µL sample was
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exposed to the X-ray beam for an average of 10-15 ms. The exposure time was controlled by the flow rate of the sample past the beam and an electronically controlled shutter. RNA folded in the absence of TP30 was exposed for 5-10 ms. Native 30S particles were exposed for 15 ms. The total extent of cleavage assessed by extension of a primer annealing at 16S position 1508 was 2040%. Samples were mixed with 5 µL precipitation buffer (3.2 M NaCl, 25 mM EDTA, 0.1 µg/µl carrier tRNA and 0.25 µg/µl glycogen), and then extracted once with an equal volume (100 µL) phenol and once with chloroform. The RNA in the aqueous layer was precipitated with 3 volumes ethanol at –20 ˚C overnight, collected by centrifugation at 14,000 rpm 30 min at 4 ˚C, dried and dissolved in 40 µL deionized water (Nanopure) and stored at –80 ˚C until further use. Primer extension. The RNA was extended with reverse transcriptase as previously described,6 with the following minor modifications. RNA (0.25-0.5 µg) was annealed at 90 ˚C for 1 min with 32P-end labeled primers (8 • 105 cpm or 0.5 pmol), which annealed after nucleotides 161, 323, 419, 540, 683, 906, 1046, 1199, 1257, 1389, 1485 in the 16S rRNA.7 Extension reactions were performed with Seikagaku AMV reverse transcriptase (Associates of Cape Cod, East Falmouth, MA) at 48 ˚C for 30 min. To detect cleavage events further from the primerannealing site or at the 3’ end of the 16S rRNA, reactions were incubated for 1h30 min with twice the amount of RNA (0.5 µg) and deoxynucleotide triphosphates. The resulting cDNA were separated on a denaturing 8% polyacrylamide sequencing gel and exposed to a Phosphorimager screen (GE Healthcare). Sanger sequencing reactions were carried out using non-irradiated rRNA as a template.
€
€
Data analysis. The counts in each band were determined using a Phosphorimager (GE Healthcare), and compared with control bands whose intensity did not change over the course of the experiment. Groups of adjacent nucleotides predicted to make the same contact (with the rRNA or with a protein) and that behaved similarly over time were clustered to increase the signal to noise average as previously described.8 The relative saturation Y of each protection was determined by normalizing the data to the maximum cleavage in the absence of TP30 ( Y = 0 ) and the minimum cleavage for that position in each data set ( Y = 1.0 ). The mean relative protection of three control reactions on native 30S ribosomes ranged from 0.7 to 1.0, depending € on fluctuations in the data. Therefore, Y ≥ 0.7 was taken to represent complete or nearly complete assembly. The fractional saturation of native Progress curves for each protected region € were fit to first order or multi-exponential rate expressions, Y = A1 + A2 [1− exp(−k 2 t)] or Y = A1[1− exp(−k1t)] + A2 [1− exp(−k 2 t)]. The precision of the observed rate constants and € amplitudes was approximately ± 50%. Structural assignment of backbone protections.€The observed protection pattern of the 16S rRNA was compared with the solvent-accessible surface area of C4’ and C5’ atoms, computed from coordinates of the structure of the E. coli 70S ribosome (2avy and 2awy),9 using the program Calc-surf.10 A sphere of radius 1.4 Å was chosen to simulate solvation by water and the solvent accessible areas were computed for each atom of the 16S rRNA using the coordinates of all atoms in the 30S subunit, or only atoms in 16S rRNA. Experimentally observed protections in the 16S rRNA were assigned as arising from RNA contacts if the predicted accessibility of the C4’ atom in the 16S rRNA alone was less than 7 Å2 (denoted by a circle in schematics); in most cases these residues had no solvent accessibility. If
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the C4’ accessibility was greater than 7 Å2 in the rRNA alone but less than 3 Å2 in the 30S subunit, the residue was classified as making a protein contact (denoted by a square in schematics). Riboses that were protected experimentally but exposed to solvent in the crystal structures are denoted with an open symbol in schematics. Backbone protections arising from direct contact with an individual protein were assigned based on the predicted accessibility of the C4’ atom in the 16S rRNA alone, contacts identified in a high resolution structure of the T. thermophilus 30S complex,11 and proximity of the protein residues to the rRNA backbone in the structures of the E. coli ribosome.9
SUPPLEMENTAL FIGURES
Figure S1. Time-resolved hydroxyl radical (•OH) footprinting was used to measure progressive changes in the structure of the 16S ribosomal RNA during assembly of 30S ribosomal subunits. The irradiation time was kept to 10 ms, to resolve structural differences over short intervals.
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Figure S2. Analysis of reconstituted 30S complexes. Ribosomal subunits reconstituted under the conditions used for footprinting reactions were tested for integrity and activity. a, Sedimentation velocity in 10-40% sucrose gradients confirmed that all of the 16S rRNA was incorporated into 30S complexes when reconstituted under the conditions of the footprinting experiments. Top, mixture (1:2) of deproteinized 16S rRNA and native 30S ribosomal subunits; bottom, reconstituted subunits. The 16S rRNA was prefolded 15 min at 42 ˚C in reconstitution buffer and assembled with TP30 for 10 min at 30 ˚C before loading on the gradient. b, Analysis of 30S complexes on composite agarose polyacrylamide gels12 shows that the reconstituted subunits have the same mobility as the native 30S minus S1, but are slightly more heterogeneous. c, Reconstituted 30S subunits were 45% as active as native 30S subunits in peptidyl transferase reactions. Native or subunits reconstituted at 30 ˚C or 42 ˚C were incubated with native 50S ribosomal subunits used in peptidyl transferase assays as previously described2 (see full Methods). Dipeptide formation was monitored by paper electrophoresis. Lane (–), no 30S subunit. Because the 30S subunits were incubated an additional 45 min at 37 ˚C during the peptidyl transferase reactions, we cannot exclude that some of the final structural changes needed to produce fully active 30S subunits require longer than 3 min at 30 ˚C to be complete.
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Figure S3. Time-dependence of individual backbone interactions. Relative saturation (Y) of each protection vs. assembly time (closed circles) was determined and fit to single or double exponential rate equations (see Methods). The extent of protection was normalized to the least cut lane or to the average protection of native 30S ribosomes (open circles). Representative progress curves are shown; fitted parameters for all positions are listed in Table S1.
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Figure S4. Detailed views of assembly kinetics in each domain. a, 3’ domain (head). The 16S rRNA ribbon is colored according to the rate of backbone protections as in Figure 2: red, ≥ 20 s-1; orange, 2 – 20 s-1; green, 0.2 – 2 s-1; blue, 0.01 – 0.2 s-1. Proteins shown as C alpha ribbons (2awy)9: S2, blue; S3, dark pink; S7, yellow; S9, seagreen; S10, cyan; S13, light pink; S14, violet; S19, orange. Left, Nomura assembly map;13,14 center, 50S interface view; right, solvent view. b, central domain (platform), as in a. Also shown in pink are H1, H44 (top), and H45. S5, green; S6, sea green; S8, orange; S11, dark pink; S15, cyan; S18, yellow; S21, violet. c, 5’ domain (body), as in a. S4, magenta; S12, orange; S16, cyan; S17, lime green; S20, yellow.
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Figure S5. Kinetics of backbone protection from protein contacts. Individual proteins protect segments of their rRNA binding site at different rates, consistent with induced fit of RNA-protein interactions. Fractional saturation of hydroxyl radical protection (Y) versus time as in Figure S3. Protections shown in each plot are attributed to direct interaction with protein S4, S7, S15 and S16, respectively. Each of these proteins contacts different segments of their rRNA binding site with different kinetics. Observed rate constants for each phase are shown (see also Table S1).
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Figure S6. Kinetics of selected protein-RNA interactions. a, S7 indirectly protects nucleotides throughout the 3’ domain. Regions of the 16S rRNA backbone protected by S7 binding were taken from Powers and Noller15 and colored according to the slowest rate constant for backbone protection, as shown in the key. b, Kinetics of S8-backbone contacts in the central domain. c, S9 rapidly protects H39. d, S16 contacts H7 and H12 in the 5’ domain of the 16S rRNA immediately, but interactions with other helices saturate at an intermediate rate, and those with H21 in the central domain form slowly. e, S16 indirectly stabilizes backbone protections throughout the 5’ domain and surrounding the central pseudoknot. Indirect S16 protections were determined as in a.
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SUPPLEMENTARY INFORMATION
doi: 10.1038/nature07298
Table S1. Observed rates of RNA backbone protection during 30S ribosome assembly at 30 ˚C.a nts 17-20 22-25 29 33-37 41-47 50-51 61-63 64-66 67-69 72-74 94 99-100 102-106 107-114 115-116 119 123-125 126-127 129 131-133 136-137 143 152 172-174 184 186-187 193-195 196-200 202 210 216 219-224 228-229 232-233 236-238 239 243-244 246-248 250-252 253 254-255 262 263 264-265 266
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helix H2 H1 H1 H3 H4 H5 H6 H6 H6 H6 H6 H6 H6 H6a H11a H11a H7 H7 H7 H7 H7 H7 H8 H8 H9 H9 H9 H9/H10 H10 H10 H10 H10/H7 H7 H7 H7 H7 H11 H11 H11 H11 H11 H11 H11 H11 H11
kobs,1 (s-1) DT DT DT DT DT DT DT DT DT DT DT DT DT DT DT DT DT DT DT DT DT DT DT DT prot 1.6 0.88 DT prot prot prot DT ≥20 s-1 ≥20 s-1 0.18 prot DT ≥20 s-1 DT DT DT ≥20 s-1 ≥20 s-1 ≥20 s-1 DT
A1 (%) 20 13 9 12 14 22 14 55 27 34 20 13 26 25 26 15 27 21 16 20 21 90 80 50
kobs,2 (s-1) 0.28 0.43 0.58 0.78 0.57 0.24 0.35 0.14 0.32 0.27 0.31 0.5 0.3 0.38 0.46 0.17 0.55 0.5 0.48 0.9 1.5
A2 (%) 53 56 61 50 57 54 54 23 55 52 53 24 58 57 55 72 63 60 60 56 43
0.14
38
0.03 0.04 0.06 0.3 0.2 0.74 0.91 0.7 1.1
34 44 33 56 65 35 41 46 44
68 55 65
80 66 59 82 34 50 43 17 13 51 46 40 38
9
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doi: 10.1038/nature07298
nts 274 275 276 278 279 281-283 289 291 293-295 296-298 301 303 305-307 309 312 315 319-321 322-325 328 331-334 335 343-345 348 352-254 356-357 363 364 371-374 375-377 378-380 383-384 387-388 389 391 392 393 397 401-402 404-406 407-408 426-430 437-441 449-452 469 481-484 485-487 495
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helix H11 H11 H11 H11 H11 H11 H12 H12 H12 H12 H12 H12 H12 H12 H6a H6a H13 H13 H13 H13 H13 H14 H14 H14/H5 H5 H5 H5 H15 H15 H15 H15 H15 H15 H15 H15 H15 H4 H4 H16 H16 H16 H17 H17 H17 H17 H17 H17
kobs,1 (s-1) 0.47 0.68 19 5.2 8.5 2.3 DT prot ≥20 s-1 ≥20 s-1 DT 0.9 1.1 ≥20 s-1 prot prot DT DT DT DT DT DT DT DT DT DT 0.6 DT DT DT DT prot DT 1.9 DT prot DT prot DT 3.1 DT ≥20 s-1 ≥20 s-1 0.75 DT DT 9.5
A1 (%) 87 78 97 87 95 68 95
kobs,2 (s-1)
A2 (%)
0.03
24
36 60 56 79 72 74
0.55
37
0.06
38
39 19 67 15 26 80 85 48 100 32 42 35 49 65-80 80
1.3 0.43
36 55
0.34 0.23
48 45
0.036
34
0.09
36
0.18 0.3
56 32
0.9
46
0.06
30
1.5 0.5 0.56
65 20 36
1.2 0.8
68 63
7 45 55 65 60 76 19 72 46 83 22 30 80
10
SUPPLEMENTARY INFORMATION
doi: 10.1038/nature07298
nts 499-502 504-505 507-509 510-511 512 513 517-518 522 524 526 528 529 530-531 536-537 540-544 546-547 548-550 552-555 556-557 559-563 565-566 569-572 574-575 577-579 580-583 585 587 597-600 606 610 617-618 620 622-623 624-626 634-636 641 643-644 646-647 652 655 657-658 663-664 666 668 673-676 678 685-689
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helix H18 H18 H18 H18 H18 H18 H18 H18 H18 H18 H18 H18 H18 H18 H18 J3/19 H3 H3 H3 560' 560' H19 J19/20 H20 H20 H20 H21 H21 H21 H21 H21 H21 H21 H21 H21 H21 H21 H21 H22 H22 H22 H22 H22 H22 H23 H23 H23
kobs,1 (s-1) ≥20 s-1 DT 0.62 DT 0.58 0.56 DT DT 0.4 prot prot 0.73 prot ≥20 s-1 DT prot DT DT prot DT DT DT ≥20 s-1 DT 2.7 6.3 1.7 ≥20 s-1 DT ≥20 s-1 7 DT 0.08 11 ≥20 s-1 DT ≥20 s-1 DT prot DT DT DT DT DT DT DT 13
kobs,2 (s-1) 0.22 0.52
A2 (%) 50 52
0.53
69
0.5 0.5
51 30
29 45
0.06 noisy
53
45 41
noisy 0.05
39
35 31 35 43 22 54 44 64 50 28 32 30 14 84 46 24 17 49 30
0.07 0.08 0.07 0.06 0.2 0.05 0.1
47 58 53 50 54 33 52
0.1 0.07 0.07 0.06 0.13
44 60 54 52 66
0.02 0.22 0.45 0.25 0.07
52 40 58 40 63
65 65 28 25 40 18 19 50
noisy 0.7 0.07 0.6 1 0.33 0.35 0.01
29 67 45 28 36 39 49
A1 (%) 43 34 72 16 74 76 16 39 58
54
11
SUPPLEMENTARY INFORMATION
doi: 10.1038/nature07298
nts 691-692 700-701 703 707 714-726 730-737 744-746 750-751 752 753-759 764-769 776 777 778-779 780-782 786-788 792 794-797 803-805 811-812 814-817 819-820 826-827 828-830 834-837 861-866 868-870 872-874 876-878 879-880 882-886 889-890 908-910 913-914 917-925 937-941 946-952 953-954 955-956 959-961 969-976 977-979 980-982 983-984 985 999 1004-1005
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helix H23b H23b H23b H23 H23/H23a H23a/H22 H22 H22 H22 H20 H24 H24 H24 H24a H24a H24a H24a H24a H24 H24 J24/25 J24/25 H25 H26 H26 H26a H26a H26a H25 H25 H19 H27 H27 H2 H2/H28 H29 H30 H30 H30 H31 H31 J31/32 J31/32 H32 H32 H33 H33b
kobs,1 (s-1) prot prot prot 17 17 13 17 17 prot 12 ≥20 s-1 not prot ≥20 s-1 ≥20 s-1 ≥20 s-1 ≥20 s-1 ≥20 s-1 ≥20 s-1 ≥20 s-1 ≥20 s-1 ≥20 s-1 12 16 13 ≥20 s-1 ≥20 s-1 ≥20 s-1 ≥20 s-1 ≥20 s-1 ≥20 s-1 ≥20 s-1 18 14 19 DT DT DT DT ≥20 s-1 ≥20 s-1 ≥20 s-1 ≥20 s-1 ≥20 s-1 16 prot DT ≥20 s-1
A1 (%)
kobs,2 (s-1)
A2 (%)
43 50 36 38 41
0.06 0.08 0.1 0.1 0.11
45 37 47 45 41
38 46
0.1 0.09
46 28
38 29 40 48 55 43 50 36 50 23 39 39 45 37 30 68 68 71 71 77 41 22 17 26 19 15 30 43 23 40 34 38
0.01 0.02 0.03 0.02 0.04 0.05 0.08 0.08 0.008 0.1 0.07
56 46 54 47 36 36 38 43 31 33 33
0.07 0.09 0.075
27 27 27
0.025 0.08 0.14 0.24 0.24 0.24 0.19 0.14 0.15 0.05 0.05 0.05
56 49 67 64 69 74 55 51 59 55 50 29
45 24
0.2 0.2
44 69
12
SUPPLEMENTARY INFORMATION
doi: 10.1038/nature07298
nts 1009 1014-1016 1024 1031 1046-1049 1052-1055 1063-1065 1072-1075 1078-1080 1081 1084-1085 1088-1090 1093 1096-1097 1106-1107 1108-1111 1117-1118 1123-1124 1125-1128 1129 1144 1147-1148 1150-1152 1157 1159 1170-1172 1179-1182 1185-1187 1188-1189 1190-1191 1192 1194 1198-1199 1200-1201 1202-1203 1205-1206 1210 1212-1213 1214-1215 1216-1218 1220-1221 1223 1224-1229 1231-1234 1236-1239 1242-1244 1248-1251
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helix H33b H33b H33b H33a H34 H34 H34 H35 H36 H36 H36 H37 H37 H37 H35 J35/38 J35/38 H38 H39 H39 H39 H39 H39 H40 H40 H40 H40 H38 H34 H34 H34 H34 H34 H34 H34 H34 H34 J34/32 H32 H32 H32 H32 H30 H30 H30 H41 H41
kobs,1 (s-1) ≥20 s-1 ≥20 s-1 ≥20 s-1 ≥20 s-1 DT DT DT DT DT DT DT DT DT DT DT DT ≥20 s-1 17 ≥20 s-1 DT nd DT DT DT DT DT ≥20 s-1 ≥20 s-1 ≥20 s-1 ≥20 s-1 DT DT DT DT ≥20 s-1 ≥20 s-1 6.7 ≥20 s-1 ≥20 s-1 ≥20 s-1 2.7 3.2 ≥20 s-1 ≥20 s-1 9.2 ≥20 s-1 ≥20 s-1
A1 (%) 49 38 50 44 60 80 60 60 50 50 50 50 27 75 53 50 55 74 52 15 68 77 43 32 25 50 49 44 42 30 12 15 12 30 33 74 47 52 54 77 71 54 49 52 46 40
kobs,2 (s-1) 0.1 0.03 0.09 0.07
A2 (%) 42 57 49 55
0.07
37
0.98
31
0.18
23
0.034 1.1
35 55
0.08 0.12 0.05 0.05 0.06 0.07 0.08 2 3 2.5 3 0.7 1.7
50 50 63 47 45 47 45 40 64 60 67 44 59
0.15 0.14 0.1
33 37 41
0.09 0.18 0.1 0.14 0.1
37 38 42 43 57
13
SUPPLEMENTARY INFORMATION
doi: 10.1038/nature07298
nts 1251 1253-1254 1260-1261 1270-1271 1277-1281 1282-1284 1289-1291 1306-1308 1309-1314 1317-1318 1319 1322-1325 1326-1327 1336 1342-1343 1344 1347 1349-1350 1351 1352-1352 1355-1356 1357-1358 1362 1365-1367 1369-1375 1376-1377 1380 1382 1386-1387 1394-1396 1398-1399 1413-1415 1433 1434 1435 1436 1438-1439 1452 1454 1456 1457-1458 1459 1465 1466 1468
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helix H41 H41 H41a H41a H41/H41a H41 H41 H42 H42 H42 H42 H42 J42/29 J42/29 H29 H29 H43 H43 H43 H43 H43 H43 H43 H43 H43 H43 H28 H28 H28 H28 H28/44 H44 H44 H44 H44 H44 H44 H44 H44 H44 H44 H44 H44 H44 H44
kobs,1 (s-1) DT 9.2 DT DT DT DT DT DT DT 3.9 DT DT DT DT DT DT DT DT DT DT DT 12 7 32 ≥20 s-1 DT DT DT DT DT DT DT DT DT DT ≥20 s-1 12 DT ≥20 s-1 ≥20 s-1 ≥20 s-1 ≥20 s-1 0.29 11 ≥20 s-1
A1 (%) 34 59 26 34 30 52 26 40 32 44 25 35 30 56 33 41 23 34 26 31 17 60 70 70 75 80 85 82 80 60 65 75 38 23 13 32 43 70 49 31 38 43 86 50 34
kobs,2 (s-1) 0.04 0.04 0.04 0.04 0.03 0.007 0.04 0.05 0.03 0.009 0.03 0.04 0.7 0.03 0.03 0.06 0.04 0.05 0.4 0.04 0.036
A2 (%) 64 41 65 51 61 45 74 54 62 39 51 53 37 31 54 46 65 57 68 63 75
noisy noisy 0.09 0.32 0.57 0.21 0.12 0.66 0.11 0.17 0.08 0.22
89 66 77 69 55 36 51 52 39 44
0.22 0.23
38 56
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a
Observed rates of 16S rRNA backbone protection from hydroxyl radical cleavage were determined as described in Methods. DT, nucleotides that were protected within the 20 ms deadtime of the experiment; nd, not quantified, usually due to reverse transcriptase pause or weak protection; prot, nucleotide protected but rate constant was not determined. The estimated uncertainty in the fitted parameters is ±50%. Folding times shorter than 50 ms could not be determined precisely and were assigned a common observed rate constant of 20 s-1.
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Table S2. Rates of RNA backbone protection from direct protein contacts at 30 ˚C. Range of kobs (s-1)a ≥ 20 – NDc
Association rate (s-1)b ND
Initial burst (%)
S3
≥ 20 – 1
9 • 10-4
33%
S4
≥ 20 – 0.1
0.21
70-80%
S5
≥ 20– 0.14
7 • 10-3
12-60%
S6/S18
17 – 0.08
0.018/0.15
18-50%
S7
≥ 20 – 0.04
ND
26-82%
S8
≥ 20 – 0.07
0.10
17-68%
S9
≥ 20 – 0.04
4 • 10-3
34-75%
S10
≥ 20 – 0.03
5 • 10-3
23-77%
S11
≥ 20 – 0.01
0.01
30%
S12
≥ 20 – 0.03
8 • 10-4
12-50%
S13
≥ 20 – 0.05
3 • 10-3
S14
≥ 20 – 0.05
1 • 10-3
30-44%
S15
≥ 20 – 0.05
0.018
40-65%
S16
≥ 20 – 0.02
0.078
10-75%
S17
6 – 0.1
0.38
15%
S19
≥ 20 – 0.03
6 • 10-3
25-55%
S20
28 – 0.08
0.38
20-55%
Protein S2
S21
c
ND
ND
a
Maximum and minimum rate constants for nucleotides predicted to be protected from hydroxyl radical cleavage by a ribosomal protein. Residues directly contacted by more than one protein or for which the backbone protection can also be explained by contacts with other parts of the 16S rRNA were excluded. More than 20% protection in the deadtime of the experiment (20 ms) was assigned a rate constant of 80 s-1. b Association rate of proteins measured by pulse-chase and mass spectrometry, taken from Ref. 16. c No data. For S3, S11, S12, S14, and S20, fast direct contacts were only 30-50% saturated in the burst phase (kobs ≥ 10 s-1); for S2 and S5, the direct contacts formed early in assembly overlap residues predicted to be buried by the tertiary structure of the rRNA and cannot be unambiguously attributed to protein binding.
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Supplemental Notes 1. 2. 3. 4. 5. 6. 7. 8.
9. 10. 11. 12. 13. 14. 15. 16.
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