Structure of EF-G ribosome complex in a pre-translocation state

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1 Structure of EF-G ribosome complex in a pre-translocation state Yun Chen, Shu Feng, Veerendra Kumar, Rya Ero, Yong-Gui Gao

Supplementary Figure 1 Supplementary Figure 1 Conformational change of the stalk regions in the 50S subunit. (a) Global change of the stalks in the 50S subunit.

2 Supplementary Figure 1 Supplementary Figure 1 Conformational change of the stalk regions in the 50S subunit. (a) Global change of the stalks in the 50S subunit. The L1 stalk (L1 rrna and L1 protein) and L11 rrna in the POST complex (EF-G trapped by fusidic acid in the ribosome) are colored gray 1. The conformational changes of the L1 stalk and L11 rrna were indicated by arrows, while the structure of the PRE was aligned on that of

3 the POST on 23S rrna. The L11 region (L11 protein and L11 rrna) is an important component of the ribosome GTPase associated center, the rearrangement of EF-G positioning on the ribosome thereby appears to induce its conformational change. (b) A close-up view of the conformational change of the L1 rrna. H76 starts to diverge at the 23S rrna base pair G2094:C2195 shown as stick model. (c) Superposition of the present (PRE complex) L1 rrna interacting with P/E trna with that in the POST complex. Coupled to the fluctuation of L1 stalk, the P/E trna is likely placed into the classical E site position, with the CCA end inclined to act as an anchor by retaining the interactions with the E site. The classical E-site trna in the POST complex is colored blue. For clarity, only L1 rrna is shown.

4 Supplementary Figure 2

5 Supplementary Figure 2 Intersubunit bridges and EF-G positioning in the PRE (ratcheted) and POST (classical) ribosome structures. The intersubunit contacts are highlighted as surface in red for both 30S and 50S subunits. (a) and (b) represent 30S subunits in POST and PRE ribosome structures, respectively. (c) and (d) represent 50S subunits, accordingly. Bridge numbering is according to the previous report by Yusupov and coworkers 2. (e) Comparison of the EF-G binding pocket in the PRE and POST (gray) complexes (aligned on 23S rrna). The RNA helices of 16S and 23S rrnas, which are involved in EF-G binding, are labeled by h and H followed by a number, respectively. The conformational change of the two complexes is indicated by arrow. (f) EF-G bound to the two complexes shown in the same view as (e).

6 Supplementary Figure 3 Supplementary Figure 3 Sequence alignment of E. coli and T. thermophilus EF-G. The switch I and II, and the G motifs (G1-G5) are indicated.

Supplementary Figure 4 Supplementary Figure 4 Electron density map showing Mg 2+ ion (a), the active site (b) and three universally conserved bases (c), as well as

7 Supplementary Figure 4 Supplementary Figure 4 Electron density map showing Mg 2+ ion (a), the active site (b) and three universally conserved bases (c), as well as conformational change of SRL (d) and domain IV of EF-G (e). (a) Residues involved in the coordination of Mg 2+ ion are also presented. (b) Coupled to the opening of the hydrophobic gate

8 (indicated by arrows), GTP becomes accessible to the catalytic His87 repositioned into its activated conformation. (c) Three universally conserved bases A1913 (H69 of 23S rrna) and A1492 and A1493 (h44 of 16S rrna) are indicated. (d) Conformational change of the SRL. The two structures were superimposed by aligning the 23S rrnas. The SRL in the POST complex is colored gray. (e) The change of the positioning of domain IV of EF-G (domain IV in the POST complex is colored gray and Gly502 of EF-G in the two complexes are presented as spheres).

9 Supplementary Figure 5

10 Supplementary Figure 5 Comparison of G domain of EF-G and the positioning of P/E trna in PRE and POST complexes. (a) Comparison of the G domain of EF-G with respect to C-terminal domain of L12 in PRE and POST complexes. The G domain of EF-G and the C-terminal domain of L12 in the PRE complex are colored violet and blue, respectively. The corresponding domains in the POST complex are colored gray. In both PRE and POST complexes, direct contact of EF-G (G domain) with the CTD of L12 were observed. (b) Superposing of the P/E trna with the classical P-site trna by aligning the 50S subunit. 16S rrna and P-site trna in the POST complex are colored gray and slate, respectively. The 30S head swiveling and body rotation are indicated by arrows. (c) A close-up view of the ASL binding pocket. Coupled to ribosome ratcheting (30S head swiveling and body rotation), the ASL of the P/E trna advances by 8.1 Å, whereas 16S rrna in the 30S head moves by Å, and 16S rrna in the neck region moves by 6-7 Å. Such unequal bilateral displacement of the classical P-site trna into the P/E positioning causes trna deformation.

11 Supplementary Table 1 Rearrangement of Intersubunit Bridges a Bridge Post-translocation b Pre-translocation c Change 30S 50S 30S 50S S13 B1a M82,D83, R93 H38 C Disrupted B1b S13 V7,G68 S13 L56,R57,R3 L5 D116,L139 L31 G17,E Disrupted S13 A72 S19 D13,L16,P42, E43, E64,V67 h33 P1012 L5 R115 L31 R48,V50,T52, A56,F59,R61 L31 K69 New B2a h44 r1407 H69 A1912 h44 r1408 H69 A1916 h44 r1409 H69 P1914 h44 r1409 H69 C h44 A1492 H69 A1913 New h44 r1494 H69 r1912 h44 r1494 Mg H69 P Mg lost - - h44 r1495 H69 r1919 New h45 rg1517 H69 r1919 B2b h24 r783 H68 P Disrupted - - h24 P784 H68 P1836 New B2c B2d B3 h24 r770...mg... H67 P1832 Mg retained h27 r899 H67 P Disrupted h24 U793...Mg... H69 P1920 h45 P1517 Mg H69 P1920 h44 A1418 H71 GC

12 B4 h44 A1483 h44 r1484 S15 S40,H53,L5 6 S15 R64,G89 H71 CG H71 r1960 h34 G715, h34 P715,P716 S15 S40,H53,L56, V60 h34 P714,P715,P 716 Similar Similar - - S15, K44 r716 New B5 h44 r1429 H62 r1703 h44 r1428 H62 r Disrupted - - h44 r1429 H62 P1704 New B6 h44 G1443 h44 r1443,r144 6 H101 P2864 L19 R118,D122 B7a h23 A702 H68 G Disrupted h23 A702 H68 A Disrupted - - h23 A702 H68 P1847 New B7b h23 P713 L2 Q166,R Disrupted B8 h24 P774 L2 K202 h24 H66 P773...Mg.. P Disrupted - - h23 r712 L2 Q164 New - - h24 P774 L2 K202 N203 New h14 h14 L19 R41 P345,P346 P345,P346 L19 E36,K35 h14 P339 L14 E9,N13 h14 L14 h14 L14 C345...Mg... S116,A118 C345 Mg V115,S h14 U343 Mg L14 S116 New - - h14 P338 L14 E9 New - - h14 P340 L14 T96 New - - h14 r345 L14 R107 New - - h14 P346 L14 R104,R107 New

13 aabbreviations:, no change (indicated interactions are maintained between post- and pre-translocation states); -, interaction absent; h and H indicates rrna 16S rrna and 23S rrna helixes, respectively; r, ribose 2'-OH interaction; P, phosphate non-bridging oxygen interaction;...mg..., interaction mediated by a magnesium bridge 3. bthe post-translocation structure refers to the T.thermophilus 70S ribosome bound with EF-G trapped in a post-translocation state (Gao et al, 2009) 1. cthe pre-translocation structure refers to the T.thermophilus 70S ribosome bound with EF-G in a GDPCP state presented in this paper.

14 Supplementary Table 2 Interactions of EF-G with Ribosome a Post-translocation b Pre-translocation c Change 23SRNA EF-G 23SRNA EF-G H95 G2656 Asp22(G) H95 G2656 Asp22(G) Asp22 Mg H89 C2658 Gln112(G) New H95 A2660 His20(G) H95 A2660 His20(G) - H95 A2660 Gln117(G) Disrupted H95 G2661 His20(G) H95 G2661 His20(G) - H95 G2661 Ile21(G) H95 G2661 Ile21(G) - H95 G2661 His87(G) New H95 A2662 Arg61(G) New H95 A2662 Ile63 (G) New H95 A2662 His87(G) New H95 G2663 Arg61(G) New H95 G2661 Glu456(III) H95 G2661 Glu456(III) - H95 G2661 Leu457(III) H95 G2661 Leu457(III) - H95 A2662 Glu456(III) New H95 A2662 Leu457(III) H95 A2662 Leu457(III) - H69 A1912 Arg499(IV) New H69 A1913 Arg499(IV) H69 A1913 Arg499(IV) - H69 A1913 Met580(IV) Disrupted H43 A1067 Asn625(V) New H43 A1067 Ile631(V) H43 A1067 Ile631(V) - H43 A1067 Leu632(V) H43 A1067 Leu632(V) - H43 A1067 Gly633(V) H43 A1067 Gly633(V) - H44 A1095 Glu614(V) New H44 A1095 Met617(V) H44 A1095 Met617(V) Stronger H44 A1095 Gly618(V) New H44 A1095 Met634(V) H44 A1095 Met634(V) - H44 A1096 Glu614(V) New H89 U2473 Gly622(V) H89 U2473 Gly622(V) - H89 U2473 Ala626(V) H89 U2473 Ala626(V) - H95 A2660 Thr657(V) H95 A2660 Thr657(V) - H95 A2660 Arg660(V) H95 A2660 Arg660(V) - H95 A2660 Ser661(V) H95 A2660 Ser661(V) - H95 A2660 Gln664(V) H95 A2660 Gln664(V) - 16SRNA EF-G 16SRNA EF-G h5 A55 Tyr321(II) h5 A55 Tyr321(II) - h5 A55 Arg354(II) New h5 G357 Arg354(II) New h5 U358 Val322(II) New h5 U358 Gly323(II) h5 U358 Gly323(II) - h5 U359 Arg324(II) h5 U359 Arg324(II) - h5 U359 Lys381(II) h5 U359 Lys381(II) - h15 U367 Tyr340(II) h15 U367 Tyr340(II) - h15 U367 Arg351(II) New h15 U368 Arg351(II) h15 U368 Arg351(II) - h15 U368 Ala353(II) New

15 h15 U368 Arg354(II) h15 U368 Arg354(II) - h15 U368 Glu365(II) New h15 C395 Tyr340(II) h15 C395 Tyr340(II) - h15 C395 Lys349(II) h15 C395 Lys349(II) - h15 G396 Lys349(II) h15 G396 Lys349(II) - h5 A360 Arg430(III) New h15 G361 Arg430(III) Disrupted h30 U955 Val533(IV) Disrupted h30 U955 Asp569(IV) Disrupted h30 U957 Val530(IV) Disrupted h31 A965 Glu574(IV) Disrupted h44 A1492 Glu579(IV) Disrupted h44 A1493 Ser578(IV) h44 A1493 Ser578(IV) - h44 A1493 Glu579(IV) Disrupted h44 A1493 Met580(IV) h44 A1493 Met580(IV) - h44 G1494 Arg499(IV) h44 G1494 Arg499(IV) - h44 G1494 Arg504(IV) New h44 U1495 Arg499(IV) h44 U1495 Arg499(IV) - h44 U1495 Thr501(IV) New h44 C1496 Thr501(IV) Disrupted S12 EF-G S12 EF-G Arg34 Gln426(III) New Arg59 Glu422(III) Disrupted Gln78 Pro444(III) New Glu79 Thr442(III) Disrupted Glu79 Thr449(III) Disrupted Glu79 Gln421(III) Disrupted His80 Thr422(III) His80 Thr442(III) - His80 Gln421(III) New His80 Ser425(III) Disrupted His80 Thr449(III) His80 Thr449(III) - S19 EF-G S19 EF-G No discussion Thr90 Glu574(IV) mrna EF-G mrna EF-G U17 Gly502(IV) Disrupted U17 Gly503(IV) Disrupted U18 Gly503(IV) Disrupted U19 Arg504(IV) Disrupted U19 Ser578(IV) Disrupted U19 Val575(IV) Disrupted U19 Ser577(IV) Disrupted trna(psite) EF-G trna(psite) EF-G A35 His573(IV) Disrupted U36 His573(IV) Disrupted U36 Gln500(IV) Disrupted U36 Thr501(IV) Disrupted U36 Gly502(IV) Disrupted U37 Thr501(IV) Disrupted

16 aabbreviations: -,interactions remaining; h, H indicate rrna helixes (h for 16SRNA and H for 23SRNA); Roman numerals in the parentheses indicate which domain the residue belongs to. bthe post-translocation structure refers to the T.thermophilus 70S ribosome bound with EF-G trapped in a post translocation state (Gao et al, 2009) 1. c The pre-translocation structure refers to the T.thermophilus 70S ribosome bound with EF-G in a GDPCP state presented in this paper. References 1. Gao, Y. G. et al. The structure of the ribosome with elongation factor G trapped in the posttranslocational state. Science 326, (2009). 2. Yusupov, M. M. et al. Crystal structure of the ribosome at 5.5 A resolution. Science 292, (2001). 3. Zhou, J., Lancaster, L., Trakhanov, S. & Noller, H. F. Crystal structure of release factor RF3 trapped in the GTP state on a rotated conformation of the ribosome. RNA 18, (2012).

Introduction to the Ribosome Overview of protein synthesis on the ribosome Prof. Anders Liljas

Introduction to the Ribosome Overview of protein synthesis on the ribosome Prof. Anders Liljas Introduction to the Ribosome Molecular Biophysics Lund University 1 A B C D E F G H I J Genome Protein aa1 aa2 aa3 aa4 aa5 aa6 aa7 aa10 aa9 aa8 aa11 aa12 aa13 a a 14 How is a polypeptide synthesized? 2