Le ribosome comme machine moléculaire

Transcription

1 Le ribosome comme machine moléculaire 1

2 70S (Thermus thermophilus) Vue du «dessus» Vue du côté externe de 50S J.H.Cate et al. (1999) Science 285, Trois sites de liaison des ARN de transfert A : aminoacyl E P P : peptidyl A E : exit 2

3 70S: ARNt (Thermus thermophilus) 30S : fidélité du décodage 50S : synthèse peptidique Extrémités anticodon J.H.Cate et al. (1999) Science 285, S 30S P A Interfaces (T.t) 50S 30S 3

4 Evidence for the A and P sites: puromycin puromycin resembles an aminoacyl-trna it was used to show that fmet-trna f Met goes to the P site Evidence for an E (exit) site Binding of trnas and aminoacyl-trnas to E. coli ribosomes mrna Species #/ribosome Location Poly(U) acetyl-phe-trna Phe 1 P or A Poly(U) Phe-tRNA Phe 2 P and A Poly(U) trna-phe 3 P, E, and A None trna Phe 1 P None Phe-tRNA Phe 0 - None acetyl-phe-trna Phe 1 P N-C-H H O trna Phe Phe-tRNA Phe acetyl-phe-trna Phe 4

5 trna:mrna:ribosome interactions T. thermophilus Yusupov et al. Science 292, 883 (2001) Platform 3D Folding of rrna for Small Subunit 3 M head 3 m - h44 Body 5

6 Bacterial 30S subunit The main purpose of the 30S is the decoding of codons and translational fidelity Main features of the crystal structures (Ramakrishnan, Yonath) : RNA is the main structural determinant; 2D domains are independently folded; Contains binding sites for r-proteins and antibiotics; Long double helix of rrna protein free found at the interface between subunits, contains the decoding site; Codon-anticodon recognition is RNA mediated; Define mrna path at the base of head; Mouvements between domains, esp head and body; 6

7 30S (Thermus thermophilus) Head Platform Head Decoding Shoulder Resolution 5.5 Å Base Body Spur Helix 44Platform W.M. Clemon et al (1999) Nature 400, trna Contacts to 16S rrna Platform Head Shoulder Decoding center Yellow = Asite Red = E-site Orange = P-site 7

8 Crystal Structure of the 30 S Ribosomal Subunit from Thermus thermophilus: Structure of the Proteins and their Interactions with 16 S RNA Ditlev E. Brodersen, William M. Clemons Jr, Andrew P. Carter Brian T. Wimberly and V. Ramakrishnan In addition to globular domains, many of the proteins have long, extended regions, either in the termini or in internal loops, which make extensive contact to the RNA. Many ribosomal proteins share similar α+β sandwich folds, but with variable topology and ways of interactions with the RNA. Ribosomal assembly : (1) the primary binders are globular proteins that bind at RNA multihelix junctions; (2)proteins with long extensions assemble later. 8

9 9

10 10

11 OB-fold 11

12 12

13 Les trois étapes de la synthèse protéique 1. Initiation (IF2 + GTP > GDP) 2. Elongation (EF-Tu + GTP > GTP) 3. Terminaison (RF-3 + GTP > GDP) Ribosome bactérien : initiation 1. ARNt f Met : structure et séquence différentes; Formylation catalysée de Met- ARNt f Met. 2. Positionnement de séquences ~10 nucléotides en amont de l AUG d initiation (séquence de Shine-Dalgarno) par interaction avec l extrémité 3 de l ARNr 16S (sous-unité 30S). 3. Interactions IF1 et IF3 avec 30S, fixation de GTP sur IF2 et complexe avec Met- ARNt f Met ; fixation de l ARNm; fixation de 50S; libération des IF et hydrolyse de GTP en GDP. 13

14 14

15 5 3 15

16 SITE OF CROSSLINK BETWEEN WOBBLE BASE IN THE ANTICODON OF trna Val AND THE UNIVERSALLY CONSERVED C1400 IN THE 16S RNA WEAVER: FIG FOOTPRINTING trna ON 16S rrna IN 30S SUBUNIT NUCLEOTIDES IN E. coli 16S RNA THAT ARE PROTECTED FROM CHEMICAL MODIFICATION BY trna BOUND TO THE 30S RIBOSOMAL SUBUNIT RED: mrna-dependent GREEN: mrna-independent BLUE: Class III sites (trna, 50S subunits, some amtibiotics) WEAVER: FIG

17 17

18 18

19 19

20 The A site 30S particle (X-ray: 3.1 Å) Helix 44 Carter et al.,nature (2000) Interaction of IF1 with the 30S subunit. (A) Close-up of the IF1 binding site, with IF1 in purple, helix 44 in cyan, the 530 loop in green, and protein S12 in orange. (B) Overview showing the position of IF1 (purple) with respect to the 30S subunit (gray). H44, 530 loop, and S12 are colored as in (A). H, head; Bo, body; N, neck; Sh, shoulder; P, platform. (C) Overview of the 30S showing helix 44, S12, and the 530 loop as in (A), but with the AP- and E-site trnas modeled as described in the text, in dark blue, orange, and yellow, respectively. A. P. Carter et al., Science 291, (2001) Published by AAAS 20

21 Conformational changes in the 30S on IF1 binding. (A) Helix 44 in the presence (cyan) and absence (yellow) of IF1, showing significant distortions of the helix. (B) Close-up showing how IF1 and S12 together can distort helix 44. The backbone moves so much that the noncanonical base pair between A1413 and G1487 is broken. (C) The same region in the absence of IF1, showing a more regular helix and the intact base pair between A1413 and G1487. (D) Surface of the 30S subunit with IF1 bound, showing regions that are protected by association with the 50S subunit in red (see text). Helix 44 and the 530 loop are also shown. Published by AAAS A. P. Carter et al., Science 291, (2001) Initiator factor IF1 Initiator trna cannot bind in the A site Initiator trna binds directly in the P site 21

22 Facteurs d élongation bactériens EF-Tu (43kD)-GTP complexe n importe quel ARNt. Changement de structure entre GTP et GDP. ~ par ribosome. EF-Ts (74 kd) facteur de libération de GDP de EF-Tu. ~ par ribosome. EF-G-GTP translocation de 3 bases de ribosome/arnm. ~ par ribosome. 22

23 23

24 Ribosome bactérien: élongation Réaction d élongation Site A ARNt-OCO- AA-NH2 + Site P ARNt-OCO-Pept ARNt-OCO-AA-NH-CO-Pept Translocation 24

25 25

26 EF-Ts stimulates displacement of GDP from an EF-Tu-GDP complex 500 units of EF-Ts 14,000 units of EF-Ts 25,000 units of EF-Ts 26