Reading the genetic code: The 3D version. Venki Ramakrishnan MRC Laboratory of Molecular Biology Cambridge, United Kingdom
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1 Reading the genetic code: The 3D version Venki Ramakrishnan MRC Laboratory of Molecular Biology Cambridge, United Kingdom
2
3 DNA The Central Dogma
4 The Central Dogma DNA mrna
5 The Central Dogma DNA mrna Protein
6 The Central Dogma DNA mrna Protein
7 Sites of protein synthesis in cells Palade, 1954
8 Sites of protein synthesis in cells Palade, 1954 Huxley & Zubay, 1960
9 Watson clip
10 Components of ribosomes From Lodish et al. Molecular Cell Biology (2000)
11 The bacterial ribosome 70S 5 30S 50S decoding site peptidyl transferase active site nascent peptide mrna 3 codon trna exit channel adapted from A. Liljas, Science 285, (1999)
12 Peter Moore
13 Peter Moore
14 Stephen White
15 Stephen White
16 Cryo-EM views of the 30S subunit Inactive Active 50S-Bound Agrawal, Lata & Frank (1995-6)
17 Starting the 30S project in Utah
18 Use of Thermus thermophilus by the Puschino group FEBS Letters (1987) 220,
19 Crystals by removing a variable component crystal drop 30S + S1 30S S1
20 Synchrotrons: Powerful x-ray sources ESRF, Grenoble, France Swiss Light Source, Villigen NSLS, Brookhaven (USA) APS, Argonne (near Chicago)
21 MAD with selenomethionine Dispersive and anomalous terms only a few electrons Works because of minimal errors from scaling and nonisomorphism However, not enough methionines in the 30S
22 Anomalous scattering from LIII edges of lanthanides f and f (electrons) Holmium used for mannose binding protein : Weis, Hendrickson et al. (1991) Energy (ev)
23 LIII edges from hexammines
24 LIII edges from hexammines Osmium hexammine binds to major groove of RNA in way that suggested many sites in ribosome
25 LIII edges from hexammines Osmium hexammine binds to major groove of RNA in way that suggested many sites in ribosome Bruce Brunschwig & Mei H. Chou
26 Double-stranded RNA helices at 5.5 Å
27 The 5.5 Å structure of the 30S subunit Clemons et al., Nature known proteins placed central domain of RNA penultimate H44 with decoding site S20
28 The 30S team Brian Wimberly Bil Clemons Ditlev Brodersen Andrew Carter Robert Morgan-Warren
29 The atomic structure of the 30S subunit Front Back (interface) Wimberly et al., Nature, 2000
30 Direct Visualization of Antibiotics Spectinomycin Paromomycin Streptomycin Pactamycin Carter et al. Nature, 2000 Brodersen et al. Cell, 2000 Tetracycline Hygromycin B
31 The Genetic Code
32 Decoding: Selection of trna trna anticodon stem-loop (ASL) cognate near-cognate non-cognate mrna codon AAG 5 UUU 3 GAG AAG 5 UUU 3 5 AAA 3 wobble position: G:U is accepted first position: G:U is rejected
33 James Ogle Induced fit on trna binding
34 Induced fit on trna binding
35 Minor groove recognition of base-pairing geometry at first two positions Ser50 (s12) C518 A1493 C518 G530 A1493 G530 C1054 Pro48 (s12) Anticodon A36 3 Codon U1 5 AAG 5 UUU3 Anticodon A35 3 Codon U2 5 AAG 5 U UU3 Anticodon G34 Codon U3 3 5 AAG 5 UU U3
36 Base pairing and fidelity: Isosteric nature of Watson-Crick pairs T7 DNA Polymerase: Doublie et al. Nature 391 (1998) T7 RNA Polymerase: Cheetham and Steitz Science 286 (1999) The ribosome: Shows that RNA contains within itself the ability to sense base pairing geometry and ensure fidelity Anticodon A36 Codon U1
37 Delivery of trna by EF-Tu, a GTPase
38 Delivery of trna by EF-Tu, a GTPase
39 The ternary complex on the ribosome: Results from cryo-em Frozen on the ribosome after GTP-hydrolysis by the antibiotic kirromycin; about 11 Å resolution Figure from Valle et al. (2002) EMBO J cf. also Stark et al. (2002) Nat Struct Biol 9,
40 : Towards high-resolution 70S structures
41 : Towards high-resolution 70S structures High quality preparations of ribosomes (Frank Murphy, Mike Tarry)
42 : Towards high-resolution 70S structures High quality preparations of ribosomes (Frank Murphy, Mike Tarry) Scale up using zonal centrifugation from protocols of Rodnina et al (Tina Daviter, Ann Kelley)
43 : Towards high-resolution 70S structures High quality preparations of ribosomes (Frank Murphy, Mike Tarry) Scale up using zonal centrifugation from protocols of Rodnina et al (Tina Daviter, Ann Kelley) Attempt to crystallize an EF-G complex (Maria Selmer, Christine Dunham)
44 : Towards high-resolution 70S structures High quality preparations of ribosomes (Frank Murphy, Mike Tarry) Scale up using zonal centrifugation from protocols of Rodnina et al (Tina Daviter, Ann Kelley) Attempt to crystallize an EF-G complex (Maria Selmer, Christine Dunham) High-resolution 70S structure with trna/mrna (Maria Selmer, Christine Dunham, Frank Murphy, Albert Weixlbaumer, Sabine Petry)
45 The 70S ribosome with mrna and trna at 2.8 Å resolution Selmer et al., Science 2006
46 : Crystal structure of the ribosome with EF-Tu
47 : Crystal structure of the ribosome with EF-Tu Deletion of strong L9 crystal contact that precludes binding of GTPase factors (Maria Selmer, Albert Weixlbaumer)
48 : Crystal structure of the ribosome with EF-Tu Deletion of strong L9 crystal contact that precludes binding of GTPase factors (Maria Selmer, Albert Weixlbaumer) Affinity purification of EF-Tu trna - ribosome complex and initial hits from crystallization trials (Frank Murphy, John Weir)
49 : Crystal structure of the ribosome with EF-Tu Deletion of strong L9 crystal contact that precludes binding of GTPase factors (Maria Selmer, Albert Weixlbaumer) Affinity purification of EF-Tu trna - ribosome complex and initial hits from crystallization trials (Frank Murphy, John Weir) Crystallization of an EF-G complex from the L9 deletion strain (Yong- Gui Gao)
50 : Crystal structure of the ribosome with EF-Tu Deletion of strong L9 crystal contact that precludes binding of GTPase factors (Maria Selmer, Albert Weixlbaumer) Affinity purification of EF-Tu trna - ribosome complex and initial hits from crystallization trials (Frank Murphy, John Weir) Crystallization of an EF-G complex from the L9 deletion strain (Yong- Gui Gao) EF-Tu: A lot of optimization of biochemistry and crystallization, ~ 1200 trays --> 1 data set from 12 crystals, to ~3.6 angstrom resolution (Martin Schmeing, Rebecca Voorhees, Ann Kelley)
51 Communication pathway between the decoding and GTPase centers
52
53
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