Synthèse protéique
La particle 50S
Le ribosome 70S
Catalyse de la formation de la
liaison peptidique
Films à trouver sur le site de
Venki Ramakrishnan à Cambridge, UK
http://alf1.mrc-lmb.cam.ac.uk/~ribo/30S/supinfo.html
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Ribosome
Cellule
Muscle: peptidyl transferase
Brains: the decoding site
2
3
Circumstantial evidence that rRNA
has a key function in translation
Genetic indications
• No single ribosomal protein required
• Antibiotic resistance mutants in rRNA
• Error frequency mutants in rRNA
• Specific nucleotides in rRNA interact with
tRNAs (compensatory mutants)
Biochemical approaches
• Specific protections of rRNA by tRNA
• Specific cross-links between rRNA and either
tRNA or mRNA
Structure of Bacterial 50S subunit
Main features:
• Can see specific features: central protuberance, stalk, ridge
• Domains are intermingled in contrast to 30S
• Deep crevice at entrance to PTase site, no protein around
• Protein exit tunnel starts at the bottom of PTase site and
goes through subunit – diameter 20 Å
• The ribosome is a ribozyme
• Catalysis occurs through proximity and orientation
•
The r-proteins of the H. marismortui 50 S subunit can be classified into six groups according
to the structural topology of their globular domains (Figure 6). The first five groups include the
antiparallel + group (L5, L6, L10e, L15e, L22, L23), the -barrel group (L2, L3, L14, L21e, and L24),
the zinc containing group (L24e, L37Ae, L37e, and L44e), the -helical group (L19e, L29, and L39e),
and the L15 group (L15 and L18e). The sixth group, the mixed + group, includes the seven remaining
proteins that are formed from mostly parallel or mixed parallel and antiparallel -sheets with additional
-helices, and do not resemble each other in any obvious way (L4, L7Ae, L13, L18, L30, L31e, and L32).
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2.4 Å resolution for the 50S
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ARNr 23S (H.
marismortui)
80 : P-loop
Active site
92 : A-loop
N.Ban et al., (2000) Science, 289, 905-920
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Science, Vol 292, Issue 5518, 883-896, 4 May 2001
Crystal Structure of the Ribosome at
5.5 Å Resolution
Marat M. Yusupov,1* Gulnara Zh. Yusupova,1* Albion Baucom,1 Kate
Lieberman,1 Thomas N. Earnest,2 J. H. D. Cate,3 Harry F. Noller1
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E-site
tRNA
P-site
5S RNA tRNA
A-site
tRNA
Yusupov et al.
Science (2001)
Kink-turn 42
Factor
Binding
Site
L1 Site
Kink-turn
77/78
A-site
finger
Front view of 50S subunit
A-site
tRNA
A-site
Finger
P-site
tRNA
Factor
Binding
E-site
tRNA
L1 Site
Kink-turn 42
5S RNA
Top view of 50S subunit
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tRNA Contacts to 23S rRNA
Yellow = Asite
Red = E-site
Orange = P-site
IV
V
9
2284-2285
Base pairings with the
-CCA end of tRNAs
2588
Interfaces
50S
P
30S
A
50S
30S
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Intersubunit Bridges 16S rRNA
Intersubunit Bridges 23S rRNA
IV
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B7a
B2a
B2b
B2c
B6
Domain IV
B5
B3
Journal of Molecular Biology
Volume 340, Issue 1 , 25 June 2004, Pages 141-177
The Roles of Ribosomal Proteins in the Structure
Assembly, and Evolution of the Large Ribosomal
Subunit*1
D. J. Klein1, P. B. Moore1, 2 and T. A. Steitz, , 1, 2, 3
12
The protein structures fall into six groups based on their topology; they
function primarily to stabilize inter-domain interactions that are necessary to
maintain the subunit’s structural integrity.
An extraordinary variety of protein–RNA interactions is observed.
Electrostatic interactions between numerous arginine and lysine residues,
particularly those in tail extensions, and the phosphate groups of the RNA
backbone mediate many protein–RNA contacts.
Base recognition occurs via both the minor groove and widened major groove
of RNA helices, as well as through hydrophobic binding pockets that capture
bulged nucleotides and through insertion of amino acid residues into
hydrophobic crevices in the RNA.
Primary binding sites on contiguous RNA are identified for 20 of the
50 S ribosomal proteins, which along with few large protein–protein
interfaces, suggest the order of assembly for some proteins and that
the protein extensions fold cooperatively with RNA.
The structure supports the hypothesis of co-transcriptional assembly,
centered around L24 in domain I.
Finally, comparing the structures and locations of the 50 S ribosomal
proteins from H. marismortui and D. radiodurans revealed striking
examples of molecular mimicry.
These comparisons illustrate that identical RNA structures can be
stabilized by unrelated proteins.
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50S: localisation of the proteins
(H. marismortui)
N.Ban et al., (2000) Science, 289, 905-920
Front view
Central
protuberance
Back view
Stalk
wing
wing
50S: structure of
proteins
(H. marismortui)
17 globular
13 partly or completely
stretch
Red :stretched
Green : globular
N.Ban et al., (2000) Science, 289, 905-920
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Science, Vol 289, Issue 5481, 920-930 , 11 August 2000
The Structural Basis of Ribosome Activity in
Peptide Bond Synthesis
Poul Nissen, Jeffrey Hansen, Nenad Ban, Peter B. Moore, Thomas A.
Steitz
Using the atomic structures of the large ribosomal subunit from Haloarcula
marismortui and its complexes with two substrate analogs, we establish that
the ribosome is a ribozyme and address the catalytic properties of its all-RNA
active site. Both substrate analogs are contacted exclusively by conserved
ribosomal RNA (rRNA) residues from domain V of 23S rRNA; there are no
protein side-chain atoms closer than about 18 angstroms to the peptide bond
being synthesized. The mechanism of peptide bond synthesis appears to
resemble the reverse of the acylation step in serine proteases, with the base
of A2486 (A2451 in Escherichia coli) playing the same general base role as
histidine-57 in chymotrypsin. The unusual pKa (where Ka is the acid dissociation
constant) required for A2486 to perform this function may derive in part from
its hydrogen bonding to G2482 (G2447 in E. coli), which also interacts with a
buried phosphate that could stabilize unusual tautomers of these two bases.
The polypeptide exit tunnel is largely formed by RNA but has significant
contributions from proteins L4, L22, and L39e, and its exit is encircled by
proteins L19, L22, L23, L24, L29, and L31e.
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The “fragment reaction”
A-site
Puromycin:
aa-tRNA
mimic
P-site
CAACCA-fmet:
P-site tRNA mimic
(35S-Met for detection)
Reaction requires 50S subunits, Mg+2, K+ , and methanol.
Affected by mutations and antibiotics just like the intact
ribosome
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Are rRNAs the site of peptidyl transferase activity?
Can rRNA alone catalyze peptide bond formation?
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Only
RNA
in
the
reaction
site
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Peptidyl
transferase:
analogues du
substrat
Site A
Site P
Nissen et al. (2000) Science,289, 920-930
Peptidyl transferase
analogues du substrat
Nissen et al. (2000) Science,289, 920-930
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Peptidyl transferase:
proteins
rRNA 23S
Domain V: red
Inhibitor:
magenta
14 proteins
interact with
domain V
Nissen et al. (2000) Science,289, 920-930
Peptidyl transferase:
No proximity
Between
Active site
And proteins
Nissen et al. (2000) Science,289, 920-930
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Peptidyl transferase:A2486
Nissen et al. (2000) Science,289, 920-930
Peptidyl transferase:charge relay
Lowering of N3 pKa of A2486 ?
Nissen et al. (2000) Science,289, 920-930
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PT
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Peptidyl transferase:
Chemical catalytic
mechanism
Or
Orientation
and
Proximity
?
The ribosome as an entropy trap
Annette Sievers, Malte Beringer , Marina V. Rodnina and Richard Wolfenden
PNAS | May 25, 2004 | vol. 101 | no. 21 | 7897-7901
To determine the effectiveness of the ribosome as a catalyst, we
compared the rate of uncatalyzed peptide bond formation, by the
reaction of the ethylene glycol ester of N-formylglycine with
Tris(hydroxymethyl)aminomethane, with the rate of peptidyl transfer
by the ribosome. Activation parameters were also determined for both
reactions, from the temperature dependence of their second-order rate
constants. In contrast with most protein enzymes, the enthalpy of
activation is slightly less favorable on the ribosome than in solution.
The 2 x 107-fold rate enhancement produced by the ribosome is
achieved entirely by lowering the entropy of activation. These results
are consistent with the view that the ribosome enhances the rate of
peptide bond formation mainly by positioning the substrates and/or
water exclusion within the active site, rather than by conventional
chemical catalysis.
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The Active Site of the Ribosome Is Composed of Two
Layers of Conserved Nucleotides with Distinct Roles in
Peptide Bond Formation and Peptide Release
Elaine M. Youngman, Julie L. Brunelle, Anna B. Kochaniak and Rachel Green
Cell 117, 589 (2004)
Peptide bond formation and peptide release are catalyzed in the active site of the
large subunit of the ribosome where universally conserved nucleotides surround
the CCA ends of the peptidyl- and aminoacyl-tRNA substrates. Here, we describe
the use of an affinity-tagging system for the purification of mutant ribosomes and
analysis of four universally conserved nucleotides in the innermost layer of the
active site: A2451, U2506, U2585, and A2602. While pre-steady-state kinetic
analysis of the peptidyl transferase activity of the mutant ribosomes reveals
substantially reduced rates of peptide bond formation using the minimal substrate
puromycin, their rates of peptide bond formation are unaffected when the
substrates are intact aminoacyl-tRNAs. These mutant ribosomes do, however,
display substantial defects in peptide release. These results reveal a view of the
catalytic center in which an inner shell of conserved nucleotides is pivotal for
peptide release, while an outer shell is responsible for promoting peptide bond
formation.
From a chemical perspective, peptide release is a more challenging
reaction than peptide bond formation because of the lower
nucleophilicity of water relative to the primary amine of an amino
acid.
…peptide bond formation on the ribosome is accomplished primarily
by simple positioning of the substrates for catalysis and does not
depend on more sophisticated catalytic mechanisms.
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Nature Structural & Molecular Biology 11, 586 - 587 (2004)
Peptide bond formation is all about proximity
Steven T Gregory & Albert E Dahlberg
• Peptide bond formation by the
ribosome is central to the
expression of genetic information,
yet its precise mechanism has
resisted elucidation for decades.
Two recent studies indicate that
substrate orientation is the sole
driving force behind the ribosomecatalyzed reaction.
Tunnel for nascent peptide
Role of 23S rRNA (in red)
Nissen et al. (2000) Science,289, 920-930
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Tunnel of nascent peptide
Interactions with chaperonines or SRP?
Nissen et al. (2000) Science,289, 920-930
Tunnel for nascent peptide
Diameter18 à 25 Å: no helical formation
Nissen et al. (2000) Science,289, 920-930
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Tunnel of nascent peptide
Nissen et al. (2000) Science,289, 920-930
Tunnel for nascent peptide
Nissen et al. (2000) Science,289, 920-930
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30S
50S
16S rRNA
(1530 nt) +
21 proteins
5S rRNA
(120 nt) +
23S rRNA
(2900 nt) +
35 proteins
Decoding &
Fidelity
Peptide bond
Catalysis
1.45x106 Daltons
0.85x106 Daltons
Tetracyclines
Aminoglycosides
300-800 Daltons
Macrolides
Lincosamides
Streptogramines
Journal of Molecular Biology
Volume 330, Issue 5 , 25 July 2003, Pages 1061-1075
Structures of Five Antibiotics Bound at the Peptidyl
Transferase Center of the Large Ribosomal Subunit
Jeffrey L. Hansen1, Peter B. Moore1, 2 and Thomas A. Steitz, , 1, 2, 3
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Anisomycin, chloramphenicol, sparsomycin, blasticidin S, and virginiamycin M
bind to sites that overlap those of either peptidyl-tRNA or aminoacyltRNA, consistent with their functioning as competitive inhibitors of
peptide bond formation.
Two hydrophobic crevices, one at the peptidyl transferase center and the other
at the entrance to the peptide exit tunnel play roles in binding these antibiotics.
Midway between these crevices, nucleotide A2103 of H. marismortui (2062
Escherichia coli) varies in its conformation and thereby contacts antibiotics
bound at either crevice.
The aromatic ring of anisomycin binds to the active-site hydrophobic
crevice, as does the aromatic ring of puromycin, while the aromatic ring
of chloramphenicol binds to the exit tunnel hydrophobic crevice.
Sparsomycin contacts primarily a P-site bound substrate, but also
extends into the active-site hydrophobic crevice.
Virginiamycin M occupies portions of both the A and P-site, and induces a
conformational change in the ribosome.
Blasticidin S base-pairs with the P-loop and thereby mimics C74
and C75 of a P-site bound tRNA.
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