The Process of Translation: Elongation and Termination

1
The Process of Translation Elongation and
Termination
1. Determining the genetic code
2. Translation elongation

• binding of aminoacylated tRNA to the A site
• formation of new peptide bond
• translocation of the peptidyl-tRNA to the P site

3. Translation termination
4. Rescuing translation when things dont work
2
Early Experiments of Nonsense Mutations (Amber
here) and Suppressor Strains
Fig. 18.28
3
Revertants Were Used To Deduce the Identity of
the Amber Codon
Revertants gave incorporation of six different
amino acids
It was easier to sequence polypeptides than DNA!
Fig. 18.29
4
Mechanism of Suppression
Mutation in tRNA allows recognition of stop
codon and incorporation of amino acid
More than one tRNA be mutated to become a
suppressor
Fig. 18.31
5
Assay For a Release Factor
Ribosome stalled with labeled peptide
Different ribosomal fractions then added to
find one that would allow release of labeled
peptide
Fig. 18.32
6
A Simpler Assay For Release Factors
fMet-tRNAfMet loaded in P site bound to AUG
Tri-nucleotide corresponding to stop codon added
Addition of ribosome fractions containing
appropriate release factor would allow release of
labeled fMet into solution
Fig. 18.33
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Structure of Release Factor Resembles tRNA
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Key Points
1. All organisms use essentially the same genetic
code three nucleotides comprise a codon, which
specifies an amino acid or a termination.
2. Translation elongation consists of cycles of
1) multi-step binding of aminoacyl tRNA in a
ternary complex with EF-Tu and GTP 2) peptidyl
transfer, catalyzed by the 50S subunit and 3)
translocation, which is greatly facilitated by
EF-G and GTP.
3. Termination is mediated by the appropriate
stop codons and release factors, which recognize
them. In the absence of termination on aberrant
RNAs, bacterial cells have evolved the tmRNA to
rescue stalled ribosomes and target the RNA and
protein for degradation.
9
Degradation (Decay, Turnover) of Eukaryotic mRNAs
mRNA turnover is important in three central
respects
1. Functions in gene expression by rapidly
changing the amount of an mRNA available for
translation
2. Functions in quality control, principally
associated with translation to ensure that mRNA
pool is translatable
3. Functions in defense against viruses
10
Degradation (Decay, Turnover) of Eukaryotic mRNAs
A. Basic processes of mRNA decay

• Deadenylation
• Exonuclease, 3-5 (exosome)
• 5-decapping
• Exonuclease, 5-3 (Xrn1p)

B. mRNA decay in quality control
11
Major Pathways of Eukaryotic mRNA Turnover
Parker and Song, Nat. Struct. Mol. Biol. (2004)
11, 121-7
12
Eukaryotic mRNA Deadenylases
Predominant deadenylation complex in yeast
contains two nucleases, Ccr4p and Pop2p
Ccr4p is highly conserved among eukaryotes,
member of ExoIII nucleases
Parker and Song, Nat. Struct. Mol. Biol. (2004)
11, 121-7
13
Ccr4p Possesses Deadenylation Activity And Is
Central to the Activity of the Complex
Conclusion based on three pieces of evidence
overexpression of Ccr4p suppresses
deadenylation defect of a pop2 deletion
isolation of Ccr4p co-purifies with deadenylase
activity, and activity is not dependent on Pop2
point mutations In the predicted active site
abolish deadenylation activity of Ccr4p
Gel shows deadenylase activity of Ccr4 and loss
of activity upon mutation of Asp713 to Ala
Tucker et al, EMBO J (2002) 21,1427-36.
14
Structure of Pop2p A Second Nuclease
Pop2 is in RNaseD family, with homology to
3-5 exo domain of DNA polymerases
Binds two metal ions and presumably uses same
chemistry as DNA exonucleases, involving
hydrolysis reaction
Pop2 also seems to stabilize complex, as Pop2
deletion gives a more severe phenotype than mutat
that ablates Pop2 activity but leaves structure
intact
Thore et al, EMBO Reports (2003) 4, 1150-5.
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PAN Complex A Second Deadenylase Complex
Activity becomes important in yeast upon
deletion of Ccr4
Also functions early in mRNA metabolism by
shortening poly(A) tail to 55-75 nt
Parker and Song, Nat. Struct. Mol. Biol. (2004)
11, 121-7
16
Initial Poly(A) Shortening May Be Coupled to
Nuclear Export
Pan3p
17
PARN Major Deadenylase Activity In Mammals?
Suggested from in vitro experiments that PARN
is principal deadenylase in mammals
Also seems to be required for deadenylation
from AU-rich instability element (ARE) and in
nonsense-mediated decay (NMD)
Absent from yeast and flies
Complexes may be differentially regulated
Flux between different mRNP states could alter
susceptibility to different deadenylase
complexes, allowing control
Parker and Song, Nat. Struct. Mol. Biol. (2004)
11, 121-7
18
After Deadenylation 3-5 Decay or Decapping
Continued decay is by exosome, a complex of
multiple exonucleases that also functions in
nuclear processing
Parker and Song, Nat. Struct. Mol. Biol. (2004)
11, 121-7
19
Structural Model and Properties of the Exosome
Thought to form ring-like structure by analogy
with related bacterial complex polynucleotide
phosphorylase (PNPase)
Seems to have a mixture of phosphorylase and
hydrolase enzymes
Complex includes Ski2p, a DEAD-box protein
Degradation is ATP-dependent
Parker and Song, Nat. Struct. Mol. Biol. (2004)
11, 121-7
20
Alternative Second Step Decapping
Dcp1/2 are highly conserved Dcp2 is catalytic
subunit
Role of Dcp1 poorly understood
Decapping achieved by hydrolysis, yielding GDP
and 5-phosphorylated mRNA, which is a good
substrate for 5-3 exonuclease
Parker and Song, Nat. Struct. Mol. Biol. (2004)
11, 121-7
21
Pathway of Decapping
1. Pab1p is removed and mRNA is deadenylated
2. Ribosomes and translation initiation apparatus
are removed
3. Accessory proteins bind (Lsm complex, PAT,
DEAD-box protein Dhh1p)
4. Other optional proteins can affect decapping
rate (e.g. PUF proteins)
5. RNAs are transported to specific cytoplasmic
foci termed P bodies
6. Decapping and subsequent degradation occur
within P body
Coller and Parker, Ann. Rev. Biochem. (2004) 73,
861-90
22
P Bodies Discrete Sites (Foci) Within Cytoplasm
Processing bodies for RNA
May also store RNAs and then later release them
Full range of functions and mechanisms of
regulation remain to be discovered
R. Parkers web page
23
Pathway of Decapping
1. Pab1p is removed and mRNA is deadenylated
2. Ribosomes and translation initiation apparatus
are removed
3. Accessory proteins bind (Lsm complex, PAT,
DEAD-box protein Dhh1p)
4. Other optional proteins can affect decapping
rate (e.g. PUF proteins)
5. RNAs are transported to specific cytoplasmic
foci termed P bodies
6. Decapping and subsequent degradation occur
within P body
Coller and Parker, Ann. Rev. Biochem. (2004) 73,
861-90
24
How Does Pab1p Inhibit Decapping?
Strong evidence indicates that inhibition from
poly(A) sequence is through binding of Pab1p
Coller and Parker, Ann. Rev. Biochem. (2004) 73,
861-90
25
Inhibition of Decapping By Bound eIF4E
TLC assay for decapping using pure proteins
Addition of eIF4E, but not functionally
compromised eIF4E, inhibits decapping
Schwartz and Parker, Mol. Cell Biol. (2000) 20,
7933-42
26
Different Complexes Can Recruit Dcp1/2
Specialized pathways include NMD and with
specific structures in 3 UTR
One example known of recruitment by RNA
structure, perhaps many more?
Convergence of regulatory pathways into common
pathway for RNA destruction
Coller and Parker, Ann. Rev. Biochem. (2004) 73,
861-90
27
Final Step Is 5-3-Exonuclease Activity of Xrn1p
Xrn1p, little known about catalytic mechanism
Mutations in eIF5A affect activity, suggesting
regulation
Drosophila homolog is developmentally regulated
Parker and Song, Nat. Struct. Mol. Biol. (2004)
11, 121-7
28
Degradation (Decay, Turnover) of Eukaryotic mRNAs
A. Basic processes of mRNA decay

• Deadenylation
• Exonuclease, 3-5 (exosome)
• 5-decapping
• Exonuclease, 5-3 (Xrn1p)

B. mRNA decay in quality control

• Nonsense mediated decay (NMD)
• Nonstop decay
• No-go decay

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Pathways of Nonsense-mediated Decay (NMD)
Nonsense-mediated decay
Decay of aberrant mRNAs with a premature
termination codon (PTC)
How can a cell determine whether a stop codon
is premature? In mammals, it uses splicing history
Exon junction complex (EJC) deposited upon
splicing
If stop codon is far upstream from last EJC
complex, the stop codon is labeled as premature
and NMD is triggered
NMD is dependent on translation, indicating the
the ribosome identifies the stop codon
First, or pioneering round of translation is
thought to be different from subsequent rounds
30
Exon Junction Complex (EJC)
Tetrameric complex contains eIF4AIII
(DEAD-box), MAGOH, MLN51, Y14, and RNA
31
Pathways of Nonsense-mediated Decay (NMD)
Yeast use a different mechanism to detect
premature termination codon
Detection relies on large distance between stop
codon and poly(A) sequence (or Pab1p), but
mechanism is unclear
Artificially tethering Pab1p just downstream
from a stop codon abolishes NMD, indicating that
Pab1 is centrally involved
32
mRNA Decay Pathway in NMD
Principally decapping and 5-3 exo in both
yeast and mammals
Here, decapping does not require deadenylation
Dcp1/2 recruited by Upf1p
33
Nonstop Decay When mRNAs Lack a Termination Codon
Analogous to process carried out by prokaryotic
tmRNA
Nonstop RNA can occur -When polyadenylation
occurs prematurely - When transcription aborts
- Upon incomplete 3-5 decay
Functions of nonstop decay 1. Recycle
ribosomes 2. Degrade RNA
Ski7p binds empty A site and recruits exosome
34
No-go mRNA Decay When Translation Stalls
Established experimentally by long hairpin
(Parker and colleagues, 2006)
Requires protein Dom34, related to release
factor
Binding of Dom34 and Hbs1 presumably recruit
endonuclease (or proposed that ribosome itself is
the endonuclease)
Decay then proceeds 3-5 by exosome (for the
5 fragment) and 5-3 by Xrn1p (for the 3
fragment)
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Key Points
1. RNA decay is critical for gene regulation, RNA
quality control, and viral defense. Here we
discussed the first two roles.
2. The basic process of RNA decay is
deadenylation followed either by 1) continued
exonuclease digestion 3 to 5 by the exosome, or
2) decapping. If decapped, the mRNA is typically
degraded 5 to 3 by Xrn1p.
3. Complex systems are present to ensure the
degradation of damaged mRNAs that are unable to
be translated accurately into protein. These
pathways include nonsense-mediated decay (NMB),
nonstop decay, and no-go decay.
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