Chapter 18 Elongation and Termination

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Chapter 18Elongation and Termination
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• Proteins are synthesized
• amino-terminal ? carboxyl- terminal
• mRNA is read in the 5 ? 3 direction

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AUG UUC

• Codons
• nonoverlapping AUG UUC
• overlapping AUG UGU GUU UUC

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AUG CAG CCA ACG

• Frameshift mutation add or delete base (X)
• Without code gaps AUX GCA GCC AAC
• With code gaps (Z) AUGZCAGZCCAZACGZ
• AUXG CAG CCA ACG

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• Codon consists of three bases

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• How do you tell which codon codes for an amino
  acid?

More than one triplet codes for an amino acid
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How does an organism cope with having multiple
codons for an amino acid?

• Multiple tRNAs for same amino acid
• Wobble hypothesis
• -first two base pairs must pair with anticodon
• -last base can wobble
• G in codon can pair with C or U in third
  position of codon
• inosine (I) can pair with C, U, or A
• -reduces number of tRNAs needed

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• The genetic code was originally thought to be
  universal across all of lifeits not!
• Evidence mitochondria, nuclear, and bacterial
  genomes

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• Likely only one origin of life
• Most changes present in mitochondria
• - dont code for as many proteins
• - more likely to have changes
• Standard code does exist
• - deviant codes likely evolved from standard code

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• Not likely code is random
• Effective at dealing with mutations
• - single-base changes usually result in shift to
  a chemically similar amino acid
• - transitions more common than transversions
• - ribosome more likely to misread first and
  third bases than second

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Elongation

• Takes place in three steps
• Ef-Tu and GTP bind an aminoacyl-tRNA to the
  ribosomal A site
• Peptidyl transferase forms a peptide bond
  between the peptide in the P site and the new
  aminoacyl-tRNA in the A site
• Ef-G and GTP translocate the peptidyl-tRNA to the
  P site

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• Proof of A and P sites based off experiments with
  antibiotic puromycin
• - resembles aminoacyl adenosine at the end of an
  aminoacyl-tRNA
• - binds to A site of ribosome
• - forms a peptide bond with P site peptide
  yielding peptidyl puromycin
• - not tightly bound to ribosome, so released
  soon ? translation aborted

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• Two sites on the ribosome can be defined
• 1) puromycin reactive
• 2) puromycin unreactive peptidyl-tRNA in the A
  site
• fMet-tRNA goes to the P site

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• Third site known as the E site (exit site)

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Step One of Elongation

• Factor T transfers aminoacyl-tRNAs to the
  ribosome
• EF-Tu unstable factor T
• EF-Ts stable factor T
• Factor G GTPase activity

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• EF-T requires GTP to bind aminoacyl-tRNA
• Polymerization requires even more GTP

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• GTP hydrolysis is not required for aminoacyl-tRNA
  binding
• GTP hydrolysis is required for the formation of
  the peptide bond

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• EF-Tu and GTP form a binary complex
• Aminoacyl-tRNA binds
• Aminoacyl-tRNA is delivered to the A site
• EF-Tu and GTP remain bound
• GTP is hydroloyzed and EF-Tu-GDP complex
  dissociates
• EF-Ts exchanges GTP for GDP

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Evidence

• EF-T and GTP bind to nitrocellulose filter when
  together
• Adding aminoacyl-tRNA causes complex to be
  released from filter
• Only aminoacyl-tRNA causes dissociation

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More Evidence
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• EF-Tu binds to GTP
• EF-Ts converts EF-Tu-GDP to EF-Tu-GTP
• - disrupts Mg2-binding center of EF-Tu
  resulting in dissociation of GDP
• - GTP can then bind to EF-Tu

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Proofreading

• Ribosome can reject incorrect aminoacyl-tRNA
• - ternary complex dissociates because it isnt
  held tightly due to mismatches between codon and
  anticondon
• - aminoacyl-tRNA dissociates for same reason
• Cell can tolerate 0.01 error rate

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• Accuracy and speed are inversely related
• Rate of hydrolysis is very important
• - if too high, not enough time for proofreading
• - if too low, translation would be too slow

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Elongation Step 2

• Peptide bond formation-
• -No elongation factors and soluble factors
  involved
• -50S ribosome peptidyl transferase activity
  forms peptide bonds

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Fig. 18.21
Traut and Monro Experiment
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Traut and Monro Experiment to distinguish the
released peptidyl t-RNA from t-RNA bound to
ribosome
Fig. 18.22
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Noller and collaborators experiment for peptidyl
transferase catalytic site
Drawback-Could not not determine exactly if its
protein or RNA since could not eliminate the
proteins .
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• 23s rRNA and proteins L2 and L3 are needed for in
  vitro peptidyl transferase activity
• 23s rRNA lies at the peptidyl transferase
  catalytic site as demonstrated by X-ray
  crystallography.

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Elongation Step-3

• Translocation-Each translocation event moves the
  mRNA one codons length,3nt,through ribosome.
• Supporting data- Peter Lengyel and colleagues
  experiment
• - Pretranslocation complex without EF-G and
  GTP-UUU ,3nt protected at the 3 end (sequencing)
• - Post translocation (EF-G and GTP)-
• UUUACU,6nt protected at the 3 end.

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Role of GTP and EF-G-

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Stopped flow kinetic experiments-
Antitranslocation antibiotics-
Viomycin,Thiostrpton GTP analogs- Unhydrolyzable
GTP analog (caged GTP), GDP
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• GTP nd EF-G are needed for translocation though
  not needed in vitro.
• GTP hydrolysis precedes translocation and
  significantly accelerates it.
• EF-G is released from ribosome for new round of
  elongation which depends upon GTP hydrolysis

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Fig. 18.26
GTPases and Translation
Generalized G-protein cycle- G-proteins- -GTP
and GDP binding proteins having intrinsic
ATPase activity -Change confirmation based upon
binding to GDP, GTP -G bound to GTP-Active
form -GAP stimulates GTPase activity
converting to GDP (inactive form) -Reactivated
by Guanine nucleotide exchange factor
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EF-Tu and EF-G structures
-EF-Tu-tRNA-GDPNP ternary complex and EF-G-GDP
binary complex three dimensional shapes have
been determined by X-ray crystallography -Two
complexes are very similar
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Fig. 18.28
Translation Termination

• Termination needs stop codons or termination
  codons
• Discovered in T4 phage
• Amber Mutation-
• Mutation that creates a stop codon which
  terminates the translation prematurely.
• Suppressed in amber suppressor strain

• Another Evidence-Brenner and colleagues
  experiment on T4
• phage head protein

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-Ambre, Ochre and Opal mutations are
termination codons UAG-Amber mutation UAA-Ochre
mutation UGA-Opal Mutation -Ochre mutation-Not
suppressed by amber suppressors but by ochre
suppressors -Cause premature termination of
translation
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Stop codon Suppression
Suppressor tRNA- have altered anticodons
that Can recognize stop codons and prevent
termination by inserting an amino acid.
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Mechanism of Suppression
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Release Factors (Assay)
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Nirenbergs Assay for release factors
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Fig. 18.34
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• RF1,RF2 and RF3 release factors are needed for
  prokaryotic transcription termination
• RF1-For UAA and UAG
• RF2-For UAA and UGA
• RF3-For RF1,RF2 binding to ribosome (GTP-
  binding protein)
• Eukaryotes-Two release factors
• eRF1- For all three termination codons
• eRF3-For release of the finished peptide
  (GTPase)

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Fig. 18.35
Dealing with Aberrant Termination
Prokaryotes deal with non-stop mRNAs by
tmRNA-mediated ribosome rescue.
Structure of Thermus thermophilus tmRNA
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Fig. 18.36
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Eukaryotes- Exosome-mediated degradation of
eukaryotic non-stop mRNA
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Premature Termination (NAS and NMD models in
Eukaryotes)
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• NMD in mammalian cells involves a downstream
  destabilizing element.
• -If the codon is far enough upstream ,it
  looks like a premature stop codon and activates
  the downstream destabilizing element to degrade
  mRNA.Needs Upf1 and Upf2.
• NAS model-
• - NAS machinery senses a stop codon in the
  middle of the reading frame
• -Splicing pattern is changed to splice out the
  premature stop codon.Needs Upf1.

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Use of stop codons to insert unusual amino acids

• Selenocysteine-A special tRNA (which recognizes
  UGA codon) is charged with serine,which is then
  converted to selenocysteine, and the
  selenocysteyl-tRNA is escorted to ribosome by a
  special EF-Tu
• Pyrrolysine-A special pyrrolysyl-tRNA synthetase
  joins preformed pyrrolysine with a special tRNA
  that has anticodon that recognizes codon UAG.

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Possttranslation(folding Nascent Proteins)
-Most newly-made polypeptides are not properly
folded and requires molecular chaperones for
proper folding -E. coli cells have a protein
called trigger factor which associates with
ribosome in such a way as to catch the nascent
polypeptide as it emerges from ribosome exit
channel -Hydrophobic regions of nascent
polypeptide are protected from inappropriate
associations until appropriate partner is
available -Archaea and eukaryotes lack trigger
factor hence uses freestanding chaperones
,which are also present in bacteria.
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Fig. 18.41
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Fig. 18.42
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Release of Ribosomes from mRNA

• Ribosomes are released from the mRNA
  spontaneously after termination
• Need ribosome cycling factor (RRF) and EF-G
• RRF resembles to tRNA and can bind to the
  ribosomes A site,but not in position taken by
  tRNA
• It collaborates with EF-G in releasing either the
  50S ribosomal subunit,or the whole ribosome,by an
  unknown mechanism