Structure of the no-go mRNA decay complex Dom34-Hbs1 bound ... · Structure of the no-go mRNA decay complex Dom34-Hbs1 bound to a stalled 80S ribosome Thomas Becker, Jean-Paul Armache,

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Structure of the no-go mRNA decay complex Dom34-Hbs1

bound to a stalled 80S ribosome

Thomas Becker, Jean-Paul Armache, Alexander Jarasch, Andreas M. Anger,

Elizabeth Villa, Heidemarie Sieber, Basma Abdel Motaal, Thorsten Mielke, Otto

Berninghausen and Roland Beckmann

Nature Structural & Molecular Biology: doi:10.1038/nsmb.2057

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Supplementary Figure 1 In vitro reconstitution and biochemical characterization of RNC-Dom34-Hbs1 complexes by sucrose density gradient centrifugation and northern blotting. (a), northern blot of truncated (DP120) and stem-loop containing DP120 mRNA (DP120-SL) as well as RNCs programmed with truncated (TR-RNC) and stem-loop DP120 mRNA (SL-RNC). As probe a DNA-oligonucleotide complementary to the 3’-region of DP120 was used. (b, c). Ribosome binding of the Dom34-Hbs1 complexes. (b), Full length Dom34-Hbs1 complex was bound to RNCs stalled by stem-loop mRNA (SL-RNC) and RNCs stalled by truncated mRNA (TR-RNC) and empty ribosomes (80S) in the presence of GDPNP and reactions were spun through a sucrose cushion. Supernatant (S) and pellet (P) fractions were analyzed by SDS-PAGE and stained with SYPRO Orange. Note that the Dom34-Hbs1 complex binds to both SL-RNCs and TR-RNCs but not to empty ribosomes (80S). Asterisks indicate contaminations. (c), Quantification of Dom34-Hbs1 binding to SL-RNCs in the presence of GDPNP (set to 100%), GTP or without a nucleotide (apo) and to empty 80S ribosomes in the presence of GDPNP. (d), Binding of Dom34-ΔN-Hbs1 complex to SL-RNC and TR-RNC. Empty 80S ribosomes (80S) were used as a control. (e), Northern blot analysis of DP120-SL mRNA and SL-RNCs after incubation with the Dom34-Hbs1 (with either GDPNP or GTP) or RNAseA. No cleavage of the DP120-SL mRNA was observed after Dom34-Hbs1 incubation, whereas RNAseA addition leads to a complete degradation of both naked and ribosome-bound DP120-SL mRNA. (f) Ribosome splitting activity of the Dom34-Hbs1 complex: SL-RNCs were incubated with a 5 fold molar excess of Dom34-Hbs1 complex in the presence of GTP or GDPNP under low magnesium conditions (2 mM). Reactions were spun through a 5-30 % sucrose gradient and UV-profiles were recorded. RNCs (blue) show a 80S and a 40S peak, which originates from co-purified initiating 43S or 48S complexes that dissociate under low magnesium concentrations. An additional 60S peak appears after incubation with Dom34-Hbs1 in the presence of both GDPNP (green) and GTP (red) indicating that SL-RNCs can be partially split.


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Supplementary Figure 2 Sorting of the yeast SL-RNC-Dom34-Hbs1 dataset. The entire dataset was sorted first for the presence of the Dom34-Hbs1 density in the canonical translation factor binding site. The Dom34-Hbs1 containing subdataset also contained P-site tRNA (green) whereas the subdataset lacking Dom34-Hbs1 did not contain tRNA and showed a ratcheted conformation for the 40S subunit (yellow). In a second step the Dom34-Hbs1 containing subdataset was further sorted for the presence of P-site tRNA. Particle numbers for subdatasets are indicated. Small images represent the reference structures, large images show structures derived from subdatasets after sorting. Densities were cut for better visualization of the P-site tRNA.


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Supplementary Figure 3 Cryo-EM reconstructions and resolution curves. Cryo-EM maps of the SL-RNC-Dom34-Hbs1 complex with (a) and without (b) tRNA in the P-site at a resolution of 9.5 Å and 9.4 Å, respectively, according to a FSC at cutoff 0.5 (6.4 Å and 6.3 Å according the 3σ criterion). (c), Cryo-EM reconstruction of the control SL-RNC complex at a resolution of 12.1 Å. (d), Cryo-EM reconstruction of the SL-RNC-Dom34-ΔN-Hbs1 complex at a resolution of 27 Å.


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Supplementary Figure 4 From the ribosome-bound Dom34-Hbs1 structure to the eRF1-eRF3 model: Comparison with available crystal structures of Dom34, Hbs1, eRF1 and eRF3. (a), Structure of ribosome-bound Dom34-Hbs1 complex. (b), Ribosome-bound Dom34, (c-d), Crystal structures of the Thermoplasma acidophilum Pelota protein (archaeal Dom34, pdb 2QI2)1 and Saccharomyces cerevisiae Dom34 (2VGM)2, (e), Aeropyrum pernix aPelota-aeEF1α complex (A3GJ)3 (f), Schizosaccharomyces pombe Dom34-Hbs1 complex (3MCA)4, (g), Ribosome-bound Hbs1, (h-k), Crystal structures of Homo sapiens eRF3 (1R5O)5 (h), S. pombe eRF1 (1DT9)6 (i), H. sapiens eRF1-eRF3 complex (3E1Y)7 (j) and S. pombe eRF1-eRF3 complex (3E2O)7 (k). (l) Model for the ribosome-bound eEF1-eRF3 complex based on the ribosome-bound Dom34-Hbs1 complex structure (a). The color code for individual domains is as in Fig.1b.


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Supplementary Figure 5 Fitting and rRNA interaction of individual Dom34 and Hbs1 domains. (a), Top: Homology model of S. cerevisiae Dom34 fitted into the cryo-EM density (transparent mesh). N=N-terminal domain in dark blue, ce=central domain in blue, C=C-terminal domain in light blue). Bottom: Fit of individual domains into isolated densities. (b), Top section: Homology models for the Hbs1 N-terminus (residues 20-84, yellow) and the Hbs1 domains G (orange; switch I region sw1 olive green), II (red) and III (dark red) fitted into isolated cryo-EM densities (transparent mesh). Bottom section: close-up views on the Hbs1 switch I region (left; olive green) and the bound GDPNP (right, magenta). (c) Schematic secondary structure of the ribosomal 5S, 18S, 5.8S and 25S rRNA and interactions with Dom34 and Hbs1. The protein-RNA interactions are highlighted in the colors for individual domains. The color code is given by the schematic representation of domain organization below.


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Supplementary Figure 6 Difference maps. (a), Cryo-EM maps for the SL-RNC-Dom34-Hbs1 complex, the control SL-RNC (middle) and the difference map (side and top views) overlaid upon the control SL-RNC map. Note that the stalk base (sb) is moved inwards upon Dom34-Hbs1 binding as observed also for eEF2 binding to 80S ribosomes8. (b), Cryo-EM maps for the SL-RNC-Dom34-Hbs1 complex low-pass filtered at 27 Å, the SL-RNC-Dom34-ΔN-Hbs1 complex at 27 Å and the difference map (side and top views) overlaid upon the RNC-SL-Dom34-ΔN-Hbs1 complex reconstruction. The 40S subunit is colored in pale yellow, 60S in gray, P-site tRNA in green, Dom34-Hbs1 density in orange and difference density in red.


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Supplementary Figure 7 Comparison of the ribosome-bound Dom34-Hbs1 complex with the ribosome bound EF-Tu ternary complex. (a), Crown views of the large 60S (left) and 50S (right) subunits and the structures of ribosome-bound Dom34-Hbs1 (left) and EF-Tu-tRNA-GDP stabilized with the antibiotic kirromycin9. (b), same as (a) but top views. The color code for individual domains is as in Supplementary Figure 5.


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Supplementary Figure 8 Secondary structure diagrams for Dom34 and Hbs1.


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Supplementary Methods

Model building for yeast Dom34, Hbs1, eRF1 and eRF3 and molecular dynamics flexible fitting (MDFF). For generation of protein homology models, the programs HHPRED10 and MODELLER11 were used. For the Dom34 model, existing crystal structures from S. cerevisiae2 and T. acidophilum1 (pdb codes 2VGM and 2QI2) were used as templates. The Dom34 loop Lβ3-β4 was modelled based on the A. pernix pelota-aEF1α X-ray structure (3AGJ)3. Hbs1-G, Hbs1-II and Hbs1-III were modeled using crystal structures of S. pombe eRF3-GDPNP5 (1R5O), S. cerevisiae eEF1A-GDPNP12 (1G7C) and ribosome-bound Thermus thermophilus EF-Tu9 (2WRN) as templates. The N-terminal domain (NTD) of Hbs1 (residues 20-84) was modelled based on the NMR structure of the Hbs1-like domain in hypothetical protein BAB28515 (1UFZ) and the N-terminal region of several mitochondrial EF-Ts (2CP9, 1AIP,1XB2). Since in the electron density of the Dom34-Hbs1 complex protein secondary structure is visible, a highly reliable initial rigid body fit for the Dom34-Hbs1 complex could be performed. For the complete Dom34 and a major part of Hbs1, the individual domains (N-, central and C-terminal domain (CTD) of Dom34, N-, G- II- and III-domains of Hbs1) were fitted as rigid bodies into the electron density. For reconnecting individual domains, hinge regions had to be adjusted manually. Based on this initial fit, we used the molecular dynamics flexible fitting (MDFF) method13,14 to interactively refine the models. The models for S. cerevisiae eRF1 (Sup45p) and eRF3 (Sup35p) were generated based on crystal structures of human eRF1 (1DT9)6 and S. pombe eRF3-GDPNP (1R5O)5. To generate the model for the ribosome-bound eRF1-eRF3 complex, the central and CTDs of eRF1 and G-, II-, and III- domains of eRF3 were superimposed on their corresponding domains in Dom34 and Hbs1. The NTD of Sup45 was positioned by a 45° rotation relative to the CTD bringing the conserved NIKS-loop close to the A-site stop codon. For the eRF1-eRF3 model in the release conformation, the eRF1 central domain was rotated by 100° around hinge residues 139-145 and 271-276 by superimposing the α-helices α5 of eRF16 and α7 of RF215 in the ribosome-bound conformation. This movement positions the GGQ motif of the eRF1 central domain near the peptidyltransferase center close to the CCA-end of the P-site tRNA. The model for the S. cerevisiae ribosome16 was used for molecular interpretation of Dom34-Hbs1-ribosome interactions.


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Supplementary Table 1 Interactions between ribosomal proteins and Dom34.

Ribosomal protein

Residue of ribosomal protein

Domain of Dom34

Residue of Dom34

SSU

rpS3 (S3P)

145-146 Dom34-N 53-54

rpS23 (S12P)

45-46 Dom34-ce 201

55-59 Dom34-N 46, 49, 58, 80

85-87 Dom34-ce 167-171, 187,

190, 194,

134 Dom34-N 11

143-145 Dom34-ce

151, 169-174

rpS31 (S27AE)

77-87 Dom34-ce

38-40, 68, 86-

92, 120

rpS30 (S30E)

1-13 Dom34-N

21, 44, 73-82,

108-114

LSU

rpL12 (L11P)

24-26 Dom34-C

311-313, 374-

379 rpL23 (L14P)

72 Dom34-ce 232


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Supplementary Table 2 Interactions between ribosomal RNA and Dom34.

Ribosomal subunit

Ribosomal RNA

S.cerevisiae rRNA

numbering

E. coli rRNA

numbering

Dom34 domain

Residue of

Dom34

h5 432 360 Dom34-ce 183

h18

564-566 517-519 Dom34-N 47-50,

82, 103-106

575-578 528-531 Dom34-N 47-52,

58, 100, 103-104

h30-h31

1179 954

Dom34-N

94 1182-1183 957-958 10-12

1187-1190 962-965 90-93

h34

1271-1274 1051-1054

Dom34-N 50-51,

89, 100-102

1427 1196 Dom34-N 50-54,

57

h28 1634-1635 1397-1398

Dom34-N 55-58

h44 1756 1493 Dom34-N 46, 113

LS

U

H43 1242 1067 Dom34-C 312, 345,

373-374

H44 1270 1095 Dom34-C 373-375

H69 2256-2257 1913-1914

Dom34-N 62, 112

H95 3029 2662 Dom34-ce 218


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Supplementary Table 3 Interactions between ribosomal proteins and Hbs1.

Ribosomal subunit

Ribosomal protein

Residue of ribosomal protein

Domain of Hbs1

Residue of Hbs1

SSU

rpS3 (S3P)

52-56 Hbs1-N

28, 31, 37-40

90-91 29-32, 35,

37 94 29 108 24 117 20-21

124-125 25

rpS23 (S12P)

109 Hbs1-II

484

130-145

Hbs1-II

408-409, 416-417, 439-442, 483-490

Hbs1-III

531-532, 574-576

rpS30 54 Hbs1-N 22

LSU rpL23 (L14P)

127-131 Hbs1-G 205-211


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Supplementary Table 4 Interactions between ribosomal RNA and Hbs1.

Ribosomal subunit

Ribosomal RNA

S.cerevisiae rRNA

numbering

E. coli rRNA

numbering Hbs1p

Residue of Hbs1p

SSU

h14 414-417 342-345 Hbs1-G 197-198,

201 h5 430-432 358-360 Hbs1-II 412-413

h15 439-440 367-368 Hbs1-II 413, 460,

463

h16

487-489 408-410 Hbs1-N

79-84

492-495 - 75-81

LSU H95

3022 2655 Hbs1-G

211, 222, 225, 354

3026-3028 2659-2661 176-177, 281, 285


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Supplementary Table 5 Interactions between Dom34 and Hbs1. Domain of

Dom34 Residue of

Dom34 Domain of

Hbs1 Residue of

Hbs1

Dom34-ce

148 Hbs1-III 526, 533

173-174 Hbs1-G 230-231,

236 174 Hbs1-II 407-409

175-180 Hbs1-G 227, 230-239, 258

176-177 Hbs1-II 409, 455-

458 183 Hbs1-II 409

184-185 Hbs1-G 230-232 188 Hbs1-G 230 192 Hbs1-G 230

216-219 Hbs1-G 231, 254-

255 250 Hbs1-G 255

252-257 Hbs1-III524-528, 533-535, 595-598,

261 Hbs1-III 597

Dom34-C

288-294 Hbs1-III517-520, 550-555,

596

299-300 Hbs1-III 520-521, 549-550

361-365 Hbs1-III 520-524,

537 380 Hbs1-III 548-549

385-386 Hbs1-III 521, 541-544,547-

550


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Supplementary references

1 Lee, H. H. et al. Structural and functional insights into Dom34, a key component

of no-go mRNA decay. Mol. Cell 27, 938-950 (2007). 2 Graille, M., Chaillet, M. & van Tilbeurgh, H. Structure of yeast Dom34: a protein

related to translation termination factor eRF1 and involved in No-Go decay. J. Biol. Chem.(2008).

3 Kobayashi, K. et al. Structural basis for mRNA surveillance by archaeal Pelota and GTP-bound EF1alpha complex. Proc. Natl. Acad. Sci. U S A 107, 17575-17579

4 Chen, L. et al. Structure of the Dom34-Hbs1 complex and implications for no-go decay. Nat. Struct. Mol. Biol. 17, 1233-1240

5 Kong, C. et al. Crystal structure and functional analysis of the eukaryotic class II release factor eRF3 from S. pombe. Mol. Cell 14, 233-245 (2004).

6 Song, H. et al. The crystal structure of human eukaryotic release factor eRF1--mechanism of stop codon recognition and peptidyl-tRNA hydrolysis. Cell 100, 311-321 (2000).

7 Cheng, Z. et al. Structural insights into eRF3 and stop codon recognition by eRF1. Genes & development 23, 1106-1118 (2009).

8 Spahn, C. M. et al. Domain movements of elongation factor eEF2 and the eukaryotic 80S ribosome facilitate tRNA translocation. Embo J. (2004).

9 Schmeing, T. M. et al. The crystal structure of the ribosome bound to EF-Tu and aminoacyl-tRNA. Science 326, 688-694 (2009).

10 Soding, J., Biegert, A. & Lupas, A. N. The HHpred interactive server for protein homology detection and structure prediction. Nucleic Acids Res. 33, W244-248 (2005).

11 Eswar, N., Eramian, D., Webb, B., Shen, M. Y. & Sali, A. Protein structure modeling with MODELLER. Methods Mol. Biol. 426, 145-159 (2008).

12 Andersen, G. R., Valente, L., Pedersen, L., Kinzy, T. G. & Nyborg, J. Crystal structures of nucleotide exchange intermediates in the eEF1A-eEF1Balpha complex. Nat. Stuct. Mol.Biol. 8, 531-534 (2001).

13 Trabuco, L. G., Villa, E., Mitra, K., Frank, J. & Schulten, K. Flexible fitting of atomic structures into electron microscopy maps using molecular dynamics. Structure 16, 673-683 (2008).

14 Phillips, J. C. et al. Scalable molecular dynamics with NAMD. J. Comput. Chem. 26, 1781-1802 (2005).

15 Weixlbaumer, A. et al. Insights into translational termination from the structure of RF2 bound to the ribosome. Science (New York, N.Y.) 322, 953-956 (2008).

16 Armache, J. P. et al. Cryo-EM structure and rRNA model of a translating eukaryotic 80S ribosome at 5.5-A resolution. Proc. Natl. Acad. Sci. U S A 107, 19748-19753


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Structure of the no-go mRNA decay complex Dom34-Hbs1 bound ... · Structure of the no-go mRNA decay complex Dom34-Hbs1 bound to a stalled 80S ribosome Thomas Becker, Jean-Paul Armache,