Protein Synthesis

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Protein Synthesis
J. Paul Simon
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Learning Objectives

• Describe the genetic code in terms of the
  following
• Degenerate
• Non-overlapping
• Without punctuation
• Almost universal
• Define codon and anticodon.
• Describe the five major stages of protein
  synthesis
• Activation of amino acids, initiation,
  elongation, termination and release, folding and
  postranslational processing.

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4. Describe the mechanism of action of puromycin.
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Proteins are synthesized in a two-stage process
1. The structural genes on DNA are transcribed
onto complimentary strands of messenger RNA
(mRNA). The nucleotide sequence of this
transcript codes for the amino acid sequence in
the protein product.
2. The processed mRNA transcript associates with
ribosomes to produce the protein originally coded
for in the DNA. Ribosomes are non-specific
protein synthesizers that produce the polypeptide
specified by the mRNA with which they associate.
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The Genetic Code
Codons are the key to the translation of genetic
information, allowing the synthesis of specific
proteins.
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The initiation codon, AUG, signals the beginning
of a polypeptide in all cells, in addition to
coding for Met residues in internal positions of
polypeptides. Three triplets (UAA, UAG, and UGA)
do not code for any standard amino acids. These
termination codons (or stop codons) normally
signal the end of polypeptide synthesis. In
general, a reading frame without a termination
codon among 50 or more codons is called an open
reading frame.
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Characteristics of the genetic code
The code is non-overlapping codons no not share
nucleotides.
There is no punctuation beginning with a start
codon (AUG) bear the 5 end of the mRNA, the
codons are read sequentially, ending with a stop
codon near the 3 end of the mRNA.
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The code is degenerate an amino acid may be
specified by more than one codon. (Redundant
might be a better word). The code is not quite
universal. Most of the very rare known
variations in the genetic code occur in
mitochondrial DNA. The code is not quite
unambiguous. In E. coli, UGA doubles as a codon
both for termination and (sometimes) for
selenocysteine.
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The code is not quite unambiguous in E. coli,
selenocysteine is introduced into formate
dehydrogenase during translation in response to
an in-frame UGA codon (normally a stop codon). A
specialized type of serine tRNA recognizes UGA
and no other codons. It is charged with serine
and then enzymatically converted to
selenocysteine prior to use at the ribosome.
The charged tRNA will not recognize just any UGA
codon some contextual signal yet to be
identified, permits the tRNA to recognize only
those sites where selenocysteine is to be
incorporated.
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(No Transcript)
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Ribosomal subunits
Ribosomal subunits are identified by their S
(Svedberg) values sedimentation coefficients
that refer to their rate of sedimentation in a
centrifuge. Sedimentation rate is effected by
shape as well as mass.
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Structure of the bacterial ribosome at
near-molecular resolution.
tRNAs
The large subunit (50S) is on the left, the small
subunit (30S) is on the right. The two
irregularly shaped subunits fit together to form
a cleft through which the mRNA passes as the
ribosome moves along the mRNA during translation.
mRNA
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General cloverleaf structure of all tRNAs
Two arms of the tRNA are critical for function.
The amino acid arm can carry a specific amino
acid esterified by its carboxyl group to the
3-hydroxyl group of the adenylate ribose residue
at the 3 end of the tRNA.
The large dots represent nucleotides. Blue lines
are base paired nucleotides.
At the end of the anticodon arm is the anticodon
loop which always contains seven unpaired
nucleotides. This is the location of the three
nucleotide anticodon.
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Three-dimensional structure of yeast tRNAPhe
deduced from x-ray diffraction analysis
The CCA sequence at the 3 end of the tRNA which
is the attachment point for phenylalanine.
Three nucleotide anticodon
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Pairing relationships of codon and anticodon
anticodon a three base sequence on tRNA that
base-pairs with the codon on mRNA
Cells must have at least one kind of tRNA for
each amino acid. At least 32 tRNAs are required
to recognize all the amino acid codons (some
recognize more than one codon).
codon a triplet of nucleotides that codes for a
specific amino acid
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A tRNA that contains an amino acid covalently
attached to its 3-end is called an
aminoacyl-tRNA. The tRNA is then said to be
charged.
Amino acids are attached by enzymes called
aminoacyl-tRNA synthetases.
Each synthetase is specific for one amino acid
and one or more corresponding tRNAs. The ability
of the synthetases to discriminated among dozens
of tRNAs has been referred to as the second
genetic code. Ten or more nucleotides may be
involved in recognition of a tRNA by its specific
aminoacyl-tRNA synthetase. Structural features
other than sequences are important for
recognition by some of the synthetases.
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Sequences on the mRNA that serve as signals for
initiation of protein synthesis in bacteria
ribosomal RNA
5
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mRNA
Shine-Dalgarno sequence
The initiating 5 AUG is guided to its correct
starting position by the Shine-Dalgarno sequence
which base pairs with a complimentary sequence
near the 3 end of the 16S RNA of the 30S
ribosomal subunit.
In eukaryotes, the initiation 5 AUG is located
by scanning the mRNA from its 5 end until the
first AUG is located, signaling the beginning of
the reading frame.
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Protein synthesis begins at the amino-terminal
end and proceeds by a stepwise addition of amino
acids to the carboxyl-terminal end of the
polypeptide.
In bacteria, the first amino acid used to
initiate protein synthesis is N-formylmethionine.
There are two separate types of tRNAs in
bacteria that are specific for methionine
tRNAMet and tRNAfMet. Both tRNAs are charged
with methionine. However the methionine on
tRNAfMet is subsequently formylated by a
transformylase. The formyl group allows
fMet-tRNAfMet to bind only at AUG initiation
sites, and prevents it from binding at an
interior AUG which code for methionine.
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Bacterial ribosomes have three sites involved in
protein synthesis the aminoacyl or A site the
peptidyl or P site the exit or E site The fMet-
tRNAfMet is the only aminoacyl-tRNA that binds
first to the P site. All other incoming
aminoacyl-tRNAs bind first to the A site and then
subsequently to the P site. The E site is the
site from which the uncharged tRNAs leave during
elongation. In eukaryotes, methionine is the
first amino acid used to initiate translation.
It is not formylated.
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Formation of the initiation complex
IF-1 and IF-3 prevent the 30S and 50S subunits
from combining prematurely
The Shine-Dalgarno sequence guides the mRNA to
position the AUG in the correct position for
initiation
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Formation of the initiation complex
fMet- tRNAfMet binds to the initiation codon in
the P site.
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Formation of initiation complex
The 30S with mRNA, fMet- tRNAfMet and associated
initiation factors combines with the 50S
ribosomal subunit.
GTP is hydrolyzed, all three initiation factors
leave. This produces a functional 70S ribosome
called the initiation complex, ready for
elongation.
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Elongation
Binding of an incoming aminoacyl-tRNA
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Elongation
Peptide bond formation
Peptide bond formation is catalyzed by peptidyl
transferase (historical name). The reaction may
be catalyzed by RNA the 23S RNA component of the
ribosome.
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Elongation
Translocation
The ribosome moves one codon toward the 3 end of
the mRNA using energy provided by the hydrolysis
of GTP. The dipeptidyl tRNA is now entirely in
the P site, leaving the A site open for the next
incoming charged tRNA. The uncharged tRNA
dissociates from the E site, and the elongation
cycle begins again.
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Termination
Termination occurs in response to a termination
codon in the A site. A release factor binds to
the A site. This leads to hydrolysis of the
ester linkage between the nascent polypeptide and
the tRNA in the P site and release of the
polypeptide. Dissociation of components.
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Coupling of transcription and translation in
bacteria
The mRNA is translated by ribosomes while it is
still being transcribed from DNA. This is
possible in bacteria because the mRNA does not
have to be transported out of the nucleus to
encounter ribosomes in the cytoplasm.
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Large clusters of 10 to 100 ribosomes that are
very active in protein synthesis can be isolated
from both bacterial and eukaryotic cells. These
clusters are called polysomes.
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Posttranslational modifications
Amino-terminal and carboxyl-terminal
modifications protease cleavage, N-acetylation
of N-terminal Loss of signal sequences by
proteolytic cleavage Modification of individual
amino acids phosphorylated ser, thr, tyr. N- and
O-linked oligosaccharides Covalently bound
prosthetic groups heme, biotin Formation of
disulfide cross-links
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Phosphorylated amino acids
The phosphate groups add negative charges to
these amino acids. The milk protein casein has
many phosphoserine groups that bind
Ca. Phosphorylation-dephosporylation cycles
regulate the activity of many enzymes and
regulatory proteins.
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Carboxylated glutamate
The blood clotting protein prothrombin contains a
number of g-carboxylglutamate residues in its
amino-terminal region introduced by an enzyme
that requires vitamin K. These carboxyl groups
bind Ca, required to initiate the clotting
mechanism.
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Some methylated amino acids
Monomethyl- and dimethyllysine residues occur in
some muscle proteins and cytochrome c. Calmodulin
of most organisms contains one trimethyllysine at
a specific position. When the carboxyl group of
glutamate is methylated, the negative charge is
removed.
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Disruption of peptide bond formation by puromycin
The antibiotic puromycin resembles the aminoacyl
end of a charged tRNA, and it can bind to the
ribosomal A site and participate in peptide bond
formation.
The product of the reaction, instead of being
translocated to the P site, dissociates from the
ribosome, causing premature chain termination.
Peptidyl puromycin
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Mechanism of action of streptomycin
Streptomycin, a basic trisaccharide, causes
misreading of the mRNA in bacteria at relatively
low concentrations. At higher concentrations, it
inhibits the initiation of translation.
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Mechanism of action of chloroamphenicol
Chloramphenicol inhibits protein synthesis in
bacteria and eukaryotic mitrochondria by blocking
peptidyl transferase. It does not effect
cytosolic protein synthesis in eukaryotes.
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Targeting of Proteins
Transport to the endoplasmic reticulum (ER) is
directed by amino-terminal signal sequences.
Human preproinsulin signal sequence
Signal sequences vary in length from 13 to 36
amino acids. They all have 10 to 15 hydrophobic
residues (yellow), one or more positively charged
residues (blue) near the N-terminal, and a short
sequence near the cleavage site that is
relatively polar with residues with short side
chains (especially ala).
The carboxyl terminal of the signal sequence is
defined by a cleavage site.
Proteins with these signal sequences are
synthesized on ribosomes attached to the ER
(rough endoplasmic reticulum).
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Directing eukaryotic proteins with the
appropriate signal sequence to the endoplasmic
reticulum
(1) The ribosomal subunits form an initiation
complex and begin protein synthesis. (2) If an
appropriate signal sequence appears, the signal
recognition particle (SRP) (3) binds to the
ribosome, binds GTP, and halts elongation. (4)
The ribosome-SRP complex binds to receptors on
the ER. (5) The SRP dissociates and is recycled,
accompanied by the hydrolysis of GTP. (6) Protein
synthesis resumes, coupled to the translocation
of the polypeptide chain into the lumen of the ER
through the peptide translocation complex. (7)
The signal sequence is cleaved by a signal
peptidase. (8) Upon termination of protein
synthesis, the ribosomal subunit dissociate and
are recycled.
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Directing eukaryotic proteins with the
appropriate signal sequence to the endoplasmic
reticulum (ER)
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Glossary
translation The process in which the genetic
information present in an mRNA molecule specifies
the sequence of amino acids during protein
synthesis. aminoacyl-tRNA An aminoacyl ester of
a tRNA. aminoacyl-tRNA synthetases enzymes that
catalyze synthesis of an aminoacyl-tRNA at the
expense of ATP energy. genetic code the set of
triplet nucleotides in DNA (or mRNA) coding for
the amino acids in proteins. codon a sequence of
three, adjacent nucleotides in a nucleic acid
that codes for a specific amino acid. anticodon
a specific sequence of three nucleotides in a
tRNA, complimentary to a codon for an amino acid
in a mRNA. reading frame a contiguous and
nonoverlapping set of three-nucleotide codons in
DNA or RNA. open reading frame a group of
contiguous nonoverlapping nucleotide codons in a
DNA or RNA molecule that do not include a
termination codon.
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initiation codon AUG (sometimes GUG in
prokaryotes) codes for the first amino acid in
a polypeptide sequence N-formylmethionine in
prokaryotes and methionine in eukaryotes. terminat
ion codon UAA, UAG, and UGA in protein
synthesis, signal the termination of a
polypeptide chain. Also know as stop
codons. mutation an inheritable change in the
nucleotide sequence of a chromosome. lethal
mutation a mutation that inactivates a
biological function essential to the life of the
cell or organism. point mutation one base in DNA
is altered, producing a change in a single codon
of mRNA. silent mutation a mutation is said to
be silent when it does not effect the amino acid
composition of the polypeptide. missense
mutation the mutation causes one amino acid to
be replaced by another. nonsense mutation
produces a stop codon which causes the premature
termination of a polypeptide chain.
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insertion mutation a mutation caused by the
insertion of one or more extra nucleotides in
DNA. deletion mutation a mutation resulting from
the deletion of one or more nucleotides from a
gene. frame shift a mutation caused by insertion
or deletion of one or more nucleotides changing
the reading frame of codons during protein
synthesis.
LAST SLIDE