ribosomes: machines that synthesize proteins · in protein biosynthesis amino acids schimmel lab...
TRANSCRIPT
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1
John Reader
Department of Cell and Developmental Biology,
University of North Carolina, at Chapel Hill
Ribosomes: Machines that
Synthesize Proteins
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Amino acids
Aminoacyl-tRNA synthetases
tRNAs
Protein Translation
Enzymes that ligate amino acids to tRNA
Essential part of the protein synthesis machinery
Amino acid specific (discriminate)
TranslationmRNA
E-TUGTP
Ribosome
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Flow of Genetic Information
Schimmel Lab
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The Genetic Code
Schimmel Lab
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Transfer RNA
3 -OH
Anticodon
Tertiary structureSecondary structureSchimmel Lab
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The ribosome is an extremely large molecular machine
Bacterial ribosomes are comprised of:
2 subunits: 50S large subunit & 30S small subunit
3 RNA molecules >4500 nucleotides in length
>50 different proteins
Molecular weight of 2.5 million daltons
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Secondary structure of large subunit ribosomal RNA from Thermus thermophilus
Noller, H. http://rna.ucsc.edu/rnacenter
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Peptidyl transferase reaction catalyzed
by the ribosome
Beringer & Rodnina (2007). Mol. Cell. 26: 311-321
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Biophysical techniques used to study
the ribosome
X-ray crystallography
Cryo-electron microscopy
smFRET
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If the ribosome requires proteins to
function, where did the proteins come
from to make the first ribosome?
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The H. marismortui large ribosomal subunit
N Ban et al. Science 2000;289:905-920
The ribosome is a ribozyme
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Structure of the peptidyl transferase center in the 50S subunit of the ribosome
Beringer & Rodnina (2007). Mol. Cell. 26: 311-321
Peptidyl- & aminoacyl-tRNA substrate analogs Transition state analog
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Beringer & Rodnina (2007). Mol. Cell. 26: 311-321
Proposed proton shuttling mechanism for the
peptidyl transferase reaction
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Polypeptide tunnel
Mankin, AS (2006) TIBS Vol 31(1):11-13
PT-peptidyl transferase center
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TM Schmeing & V Ramakrishnan (2009) Nature 461, 1234-42
Complete structure of the ribosome
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Translation elongation cycle
Frank, J. http://www.columbia.edu/cu/franklab/index.html
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Molecular mimicry by Elongation factors
EF-G EF-TUGTP*
+ aminoacyl-tRNA
Neirhaus, K. & Wilson D (2005) Enc. of Life Sciences:1-10
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Ribosome Translocation- a ratchet mechanism involving EF-G
Cornish, P et al., (2008) Mol. Cell. 30(5): 578-588
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The Nobel Prize in Chemistry 2009
“for studies of the structure and function of the ribosome”.
Nobelprize.org. 2 Jun 2011 http://nobelprize.org/nobel_prizes/chemistry/laureates/2009/
Tom Steitz Ada YonathVenki Ramakrishnan
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Big questions in protein translation
What is the origin of the ribosome ?
How did the genetic code evolve ?
Which amino acids came first in proteins ?
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tRNAs
Aminoacyl-tRNA
synthetases
Aminoacyl-tRNAs
Proper translation
of the genetic code
Role of Aminoacyl-tRNA Synthetases
in Protein Biosynthesis
Amino acids
Schimmel Lab
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(1) E + AA + ATP E(AA-AMP) + PPi
(2) E(AA-AMP) + tRNA E + AA-tRNA + AMP
The 20 aminoacyl-tRNA synthetases all share a common mechanism
+ ATP
+ AMP + PPi
AA’s Conserved catalytic domain
Anti-codon binding domain
Cognate tRNA
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24
Reaction Catalyzed by AARSs
O
OHO
HH
H
CH2
H
OPO
O
O
Adenine
tRNA
C
HC
O
NH3+
R
Aminoacyl-tRNA
(terminal 3’nucleotide
of appropriate tRNA)
3’ 2’
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SerRS
ThrRS
AlaRS
GlyRS
ProRS
HisRS
AspRS
AsnRS
LysRS
PheRS
MetRS
ValRS
LeuRS
IleRS
CysRS
ArgRS
GluRS
GlnRS
TyrRS
TrpRS
Class I Class II
Aminoacylates
tRNA 2 OHAminoacylates
tRNA 3 OH
Aminoacyl-tRNA synthetases enzymes can
be divided into 2 different classes
Ribas de Pouplana & Schimmel (2001)
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tRNAs
Mischarged
tRNAs
Aminoacyl-tRNA
synthetases
Aminoacyl-tRNAs
Proper translation
of the genetic code
Errors in
protein synthesis
Role of Aminoacyl-tRNA Synthetases in Protein Biosynthesis
Amino acids
+Editing
Schimmel Lab
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Protein Translation Studies: practical
applications
Development of Antibiotics
Expanding the genetic code
OH
OH2N
OH
OH
OH2N
OH
OH2N
OH
OH2N
O
OH
OH2N
N3
O O O
P.G. Schultz, TSRI
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Infected plant cell
Pathogenic Agrobacterium tumefaciens
Agrocinopine
Agrobacterium radiobacter (Biocontrol)
Mullins et al. 2001
Pathogenic Agrobacterium tumefaciens causes crown
gall tumors in plants
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A. radiobacter (plant biocontrol agent) produces Agrocin
84 to compete with A. tumefaciens (pathogen)
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TM84 is a potent inhibitor of leucyl tRNA synthetases (LeuRSs)
0
0.2
0.4
0.6
0.8
1
0 5 10 15 20
Fra
cti
on
al
am
ino
ac
yla
tio
n
ac
tiv
ity
(v
i/v0
)
[TM84] nM
Kiapp = 0.26 ± 0.13 nM
1) Aminoacyl adenylate formation:
LeuRS + Leu + ATP LeuRS (Leu-AMP) + PPi
2) Aminoacyl transfer:
LeuRS (Leu-AMP) + tRNALeu LeuRS + Leu-tRNALeu + AMP
LeuRS Reaction:
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TM84 (Toxic Moiety 84) Leu-AMP
Agrocin 84
TM84 closely resembles Leu-AMP
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Hypothesis: TM84 binds acts as a stable Leu-AMP mimic to inhibit aminoacylation
IC50 = 25 µM
IC50 = 1 nM
tRNALeu is essential for the tight-binding of TM84!
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Utilizing Isothermal Titration Calorimetry (ITC) to dissect the mechanism of inhibition by TM84:
Unbound ligand Bound ligand
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E E• TM84
Kd = 152 nM
E • tRNA E• tRNA • TM84
Kd1 = 0.8 nMKd2 = 14.4 nM
tRNALeu is essential for tight-binding of TM84
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TM84 binds to E. coli LeuRS-tRNALeu in theaminoacylation-like conformation!
Editing domain
tRNAleu
Catalytic domain
Anticodon-binding domain
TM84
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