Transcript Translation

Translation
Chapter 9
Overview
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Occurs on ribosomes-large aggregates of
rRNA and protein
tRNA acts as amino acid carriers
Prokaryotes—occurs simultaneously with
transcription and mRNA degradation
Eukaryotes—occurs in cytoplasm
mRNA translated 5’3’
Protein synthesis aminocarboxy
Protein Synthesis
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Polymerization of amino acids:
condensation reaction (dehydration
synthesis)
~Universal Genetic Code
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Codons—sets of 3 nucleotides corresponding to
a single amino acid
Each codon specifies a single amino acid
More than one codon can specify the same
amino acid
code is said to be degenerate
Some aa correspond to a single codon
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AUG—initiator codon, methionine (Met, M)
UGG–Tryoptophan (TrP, W)
Often codons encoding the same aa differ onl;y
at the 3rd nucleotide
~Universal Genetic Code
Why~Universal? Exceptions
GUG sometimes used as a start
 Mammalian mitochondria
NH
 Ciliated protozoa
 Selenocysteine
H-C-CH Se

3
+
2
COO-
Selenocysteine
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The 21st amino acid?
An essential amino acid for selenoproteins EX.
Glutathione oxidase
Uses unique tRNA (tRNASec), initially bound to
Ser. Longest known tRNA (95nt).
Anticodon recognizes UGA (Stop) as Sec
Signal (a stem loop configuration) 3’ to the UGA
determine Stop or Sec
Dedicated specific elongation factor recognizes
the stem-loop and substitutes for usual
elongation factor (EF-Tu)
Degeneracy—Wobble Hypothesis
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Explains how some tRNA recognize more than
one codons
tRNA molecules only need to make strong base
pairs with 2 of the three codons in the
nucleotide
This third loose base pairing interaction is called
wobble
Note: only certain bases can substitute for
others
Wobble Example
This UCA codon
was read by the
tRNA with a
UGA anticodon
But if this UCA was
UCG, it would still
have been read by
the tRNA with a UGA
anticodon
Codon Usage
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More than one codon exists for most amino acids
(except Met and Trp)
Organism may have a preferred codon for a
particular amino acid
Codon usage correlates with abundance of tRNAs
(preferred codons are represented by abundant
tRNAs)
Rare tRNAs correspond to rarely used codons
mRNAs containing rare codons experience slow
translation
Implications for GE?
Amino Acyl Synthetase and tRNA
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Amino acyl synthetases catalyze
attachment of aa to its appropriate tRNA
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One for each amino acid
tRNA
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Derived from large 1º transcript
Heavily modified, unusual bases
Extensive folding due to internal H-bonding
Amino Acyl
Synthetase
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Carboxy end of aa
attached to -phospate
of ATP
AMP released as carboxy
end of amoinoacyl group
transferred to O at C-3
of 3’nt
When aa is attached,
tRNA is charged or
acylated
No aa = uncharged
Wrong aa = mischarged
NOTE PPi
tRNA Activation by Aminoacyl tRNA Synthetases
O
1. Aminoacyl-AMP formation:
HO
(-)O
O
P
R
+H3N
O
O(-)
P
O
R
O(-)
+H3N
O
C
P
O
O-
Adenine
O
O
C
O
O
P
O
O
Adenine
O
O-
OH OH
Aminoacyl adenylate
(Aminoacyl-AMP)
OH OH
+
PPi
2Pi
2. Aminoacyl transfer to the appropriate tRNA:
R
+H3N
R
C
O
O
O
P
O
O-
Adenine
O
+
HO-ACC-tRNA
+H3N
C
O
ACC-tRNA
+ AMP
O
OH OH
Overall reaction: amino acid + tRNA + ATP  aminoacyl-tRNA + AMP + PPi
tRNA Function and Structure
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Anticodoncomplementary to
codon on mRNA
Amino attachment
(CCA) site
Other recognition
sites
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DHU loop
TC Loop
Extra arm (variable)
NOTE: also unusual
bases observed
acceptor stem
acceptor stem
tRNA Recognition by Amino Acyl
Synthetase
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Sequence elements in each tRNA are
recognized by its specific synthetase
including:
One or more bases in acceptor stem
 Base at position 73 “Discriminator base”
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 Seems
to play a major role in many cases, but
in other cases it is completely ignored.
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In many, at least one anticodon base
Recognition (cont’d)
No common set of rules for tRNA recognition !!!
 Anticodon region is not the only recognition
site
 The "inside of the L" and other regions of the
tRNA molecule are also important
 Specificity of several aminoacyl-tRNA
synthetases determined by:
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one or more bases in anticodon
one or more bases in the acceptor stem
discriminator base 73
Mischarging
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Observation: several aa similar in size and shape,
but mischarging rare.
Editing carried out by aminoacyl tRNA synthetase
Ex Double sieve of isoleucine synthetase
 Activation site– coarse sieve, rejects aa larger
than ile. excluded because they don’t fit.
 Editing (hydrolytic) site—fine sieve. Accepts
activated amino acids that are smaller than ile
(ex, Val-AMP), but rejects Ile-AMP (too large).
those that get through are hydrolyzed to aa
and AMP. Reduces mischarging from 1/225
(expected) to 1/180,000 (observed).
Sites can also distinguish based on hydrophobicity
Isoleucil-tRNA Synthetase: Proofreading
Based on Size
Larger
Acylation Site
Larger
Acylation Site
Smaller
Hydrolytic Site
Smaller
Hydrolytic Site
CH 3
H3C
CH 3
CH 3
O
O
NH 3 +
+H 3 N
tRNAIle
O
CH 3
O
tRNAIle
Difference in Size
H 3C
CH 3
O
O
+H 3 N
+H 3 N
O
CH 3
O
tRNAIle
Ile
Correct Acylation
Val
Misacylation
tRNAIle
Valyl tRNAVal Synthetase Proofreading:
Hydrophobic/Polar Recognition Motif
Hydrophobic
Acylation Site
3 HC
Polar
Hydrolytic Site
Hydrophobic
Acylation Site
Polar
Hydrolytic Site
CH 3
H 3C
O
OH
O
+H 3 N
NH 3 +
O
tRNAVal
tRNAVal
O
Difference in Hydrophobicity
CH3
CH 3
HO
CH 3
O
O
+H 3 N
+H 3 N
O
tRNAVal
Val
Correct Acylation
O
tRNAVal
Thr
Misacylation
Experiment (1962)
tRNA-ACA
Cell-free extract
amino acids & enymes
tRNA is charged with
Cys
Cys-tRNA-ACA
Treat w metal catalyst
removes thiol groups
Anticodon (recognizes
UGU codon, encodes Cys)
RNA template
UGUGUGUGUG...
Protein
has Cys
Charged amino acid is
changed chemically
Ala-tRNA-ACA
RNA template
UGUGUGUGUG...
Protein
has Ala
Once an aminoacyl-tRNA has been synthesized the amino acid
part makes no contribution to accurate translation of the mRNA.
Protein Synthesis-3 Stages
 Initiation
 Elongation
 Termination
Ribosomes
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Composition of
eukaryotic and
prokaryotic ribosomes
Mol. Biol. Gene, Fig. 14-13
Composition
of the E. coli
Ribosome
50S subunit
23S & 5S
RNA + 34
proteins
30S subunit
16S RNA +
21 proteins
Gross
anatomy
of the E.
coli
ribosome.
Fig. 19.5
head
platform
stalk
platform
ridge
Central protuberance
stalk
Initiation
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In both prokaryotes and
eukaryotes, protein synthesis
begins with a specific initiating
tRNA
In prokaryotes, initiator
methionine amino group is
methylated
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Attached to special tRNA
(fMet-tRNA)
Transformylase—adds formyl
group
Deformylase—removes formyl
group from Met of completed
peptide
Formylation does not occur in
eukaryotes
Steps in Initiation
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Association of 30S subunit with
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mRNA
fMet-tRNA
Initiation factors (3 proteins)
GTP
}
30S Preinitiation
Complex
QUESTION: AUG encodes fMet and Met.
How does 30S ribosome “know” which aa
is to be inserted?
Initiator Codon Recognition
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fMet-tRNA responds only to initiator
codons (AUG, GUG, UUG [rarely])
Met-tRNA responds only to internal AUG
Meaning of codons dependent on their
context, i.e., sequences nearby
In Eukaryotes: 5’ cap involvement
In Prokaryotes: Shine-Dalgarno Sequence
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mRNA- (5’)AGGAG (3’)
16S rRNA- (3’)UCCUC(5’)
Shine-Dalgarno Interaction
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Upstream
from initiator
AUG
Complement
ary to a
stretch on
16S rRNA
Seen in
virtually all
prokaryotic
mRNA
Initiation Factors in Protein Synthesis
 IF-1
 Promotes dissociation of ribosome.
 IF-1 also blocks the A site of the small ribosomal
subunit
 insures the initiation aa-tRNA fMet-tRNAfMet
can bind only in the P site & that no other aatRNA can bind in the A site during initiation.
 IF-2
 small GTP-binding protein (a GTPase).
 Interacts with
 Small subunit
 IF1
 fMet-tRNAfMet
Initiation Factors in Protein Synthesis
 IF-2 (cont’d)
 IF-2/GTP helps the initiator helps it dock with the
small ribosome subunit, prevents other tRNAs from
binding small subunit
 IF-3
 Binds small subunit, prevents reassociation w/
large subunit
 binds mRNA to the 30S ribosomal subunit
 frees it from its complex with the 50S subunit.
30S Pre-initiation Complex
70S Initiation Complex Assembly
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30S pre-initiation= 30S subunit,IF1-3, mRNA, GTP, fMettRNAfMet
When fMet-tRNAfMet pairs with initiator codon, small
subunit undergoes conformational change
Result: Release of IF-3
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Binding of large subunit stimulates GTPAse activity of IF2/GTP
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Large subunit can bind small subunit complex
hydrolysis of GTP to GDP
IF-2/GDP and IF-1 fall off
RESULT: 70S ribosome with fMet-tRNAfMet in P-site
of ribosome
RESULT:
70S
Initiation
Complex
Overview
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Dissociation of inactive
70S
IF-1, IF-3
IF-2/tRNA, mRNA
fMet-tRNAfMet/initiator
codon-releases IF-3
GTP Hydrolysis, releases
IF-1, IF-2
Complete 70S complex
70S Ribosome
A (aminoacyl) site
Small
subunit
Transfer RNAs
P (peptidyl)
site
Large subunit
(50 S)
5’ end
Messenger RNA
Alternative View
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A=Aminoacyl
site
P=peptidyl
site
E=Exit site
Elongation Overview
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Aminoacyl tRNA complementary to codon in Asite moves into A-site
N-formyl-Met transferred from tRNAfMet to
aminoacyl-tRNA in A-site
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tRNAfMet leaves P-site
Ribosome moves along mRNA (translocation)
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now have a dipeptide in the A-site
dipeptide in P-site
New aminoacyl-tRNA moves into A site, etc
Elongation
ELONGATION
Transpeptidation Reaction
tRNA
P site
O
O
O
P
O
A site
tRNA
O CH2

H
Carboxy
end of
nascent
peptide
O
O
O
P
H
O
H
OH
H
O
C
R
NH
C
HC
O CH2
O
H
HC
O
Adenine
R
NH3+
O
H
H
O
H
OH
C
HC
Nucleophilic attack
Adenine
R
:NH2
Amino terminus of
incoming amino acid
Transpeptidation Completed
tRNA
The nascent
polypeptide,
one residue
longer, is
now linked
to the tRNA
in the A site.
P site
O
O
O
P
O
A site
tRNA
O CH2

H
Adenine
O
H
H
OH
H
OH
O
P
O
O CH2

H
O
O
H
H
O
H
OH
C
HC
R
NH
O
C
HC
R
NH
O
Adenine
C
HC
R
NH3+
EF-GGTP
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Role in
translocation
Binding site
uncovered
EF-G-GTP
occupies
Hydrolysis
GDP leaves
open A-site
Translation
Termination
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“Stop” Codon
No anticodons, but
are release factors
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Proteins
Occupy A-site
Activate hydrolysis
of peptide from
peptidyl-tRNA
Translation
Termination 2
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Release factors
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RF-1 and RF-2
recognize stop codons
RF-3 –stimulates
dissociation of 70S
ribosome after release
of polypeptide chain
Anticodon recognition
determined 3 aa
Proofreading in
Translation
1. Codon:anticodon
base pairing
2. 16S rRNA forms Hbonds with minor
groove of
codon:anticodon
duplex only when
correctly paired
3. Proofreading
in Translation
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Correct base pairing
allows EF-Tu bound to
aa-tRNA to interact with
factor binding center,
inducing GTP hydrolysis
and EF-Tu release
Incorrect base pairing
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FBC not contacted
allows more time for EFTu GTP release
4. Proofreading
in Translation
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Incorrectly
paired tRNA
can’t rotate into
position for
peptide bond
formation
“tRNA
accommodation”
Antibiotics
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Translation the target of many antibiotics. EX
Site of
Nucleophilic
attack
Absent
terminates
translation
Eukaryotic Translation Factors
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designated with the prefix "e"
EF-Ts replaced by
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eEF-1
eEF-2 (target for diphtheria toxin)
Diphtheria Toxin

EF2 is only known substrate for diphtheria toxin
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Addition of ADP-ribose inactivates EF2
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EF2 contains rare modification of one of histidine
residues and this is site recognized by toxin
Mutant cells that cannot modify site are resistant
Kills cells by irreversible block of protein synthesis
P. aeruginosa exotoxin A works same as
diphtheria toxin