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Chapter 6
Gene Expression:
Translation
Copyright © 2010 Pearson Education Inc.
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Chemical Structure of Proteins
◦ Proteins are built from amino acids held together by
peptide bonds. The amino acids confer shape and
properties to the protein.
◦ Two or more polypeptide chains may associate to form
a protein complex. Each cell type has characteristic
proteins associated with its function.
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All amino acids (except proline) have a common structure.
The a-carbon is bonded to:
-An amino group (NH2), which is usually charged at cellular
pH (NH3+).
-A carboxyl group (COOH),
which is also usually charged
at cellular pH (COO-).
A hydrogen atom (H).
-An R group, which is
different for each amino acid
and confers distinctive
properties. The R groups in an
amino acid chain give
polypeptides their structural
and functional properties.
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Polypeptides are chains of amino acids joined by
covalent peptide bonds. A peptide bond forms
between the carboxyl group of one amino acid
and the amino group of another
Polypeptides are un-branched and have a free
amino group at one end (the N terminus) and a
carboxyl group at the other (the C terminus).
The N-terminal end defines the beginning of the
polypeptide.
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Proteins have up to four levels of
organization:
◦ Primary structure is the amino acid
sequence of the polypeptide. This is
determined by the nucleotide
sequence of the corresponding gene.
◦ Secondary structure is folding and
twisting of regions within a
polypeptide, resulting from
electrostatic attractions and/or
hydrogen bonding. Common
examples are the a-helix and bpleated sheet.
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Tertiary structure is the threedimensional shape of a single
polypeptide chain,
conformation.
◦ It is the biologically functional form
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Quaternary structure occurs in
multi-subunit proteins, as a
result of the association of
polypeptide chains.
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4 nucleotides code for 20 amino acids; How?
◦ Combinations
◦ How many combinations?
◦ How is done?
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Evidence for a triplet code came from
experiments in bacteriophage T4. A virulent
phage, T4 produces 100–200 progeny phage per
infected E. coli cell and produces plaques on a
“lawn” of E. coli.
A mutant T4 phage strain called rII can be
identified in two ways:
◦ a. T4 phage rII mutants produce clear plaques when
grown on E. coli strain B, while the wild-type r+
phage make turbid plaques on E. coli B.
◦ b.T4 phage rII mutants do not grow in E. coli strain
K-12(l), while r+ T4 phage do.
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The rII mutant strain was
produced by treating r+ phage
with proflavin. Proflavin causes
frameshift mutations by inserting
or deleting base pairs of DNA.
The reasoning was that reversion
of a deletion (a -mutation) could
be caused by a nearby insertion (a
+mutation), and vice versa.
Revertants of rII to r+ can be
detected by plaques on E. coli K12(l).
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Combine genetically distinct rII mutants of
the same type (either all - or all +), and only
when it is a combination of three (or multiple
of three) are there high levels of reversion.
This indicates that the genetic code is a
triplet code.
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Nirenberg and Khorana (1968) using cell-free, proteinsynthesizing systems from E. coli that included ribosomes
and required protein factors, along with tRNAs carrying
radiolabeled amino acids.
Synthetic mRNAs were used in the cell-free translation
system, and the resulting polypeptides analyzed:
◦ a. When the mRNA contained one type of base, the results
were clear [e.g., poly(U) was responsible for a chain of
phenylalanines].
◦ b. Synthetic random copolymers of mRNA (a mix of two
different nucleotides, A and C for example) can contain eight
possible codons, including two with only one nucleotide (e.g.,
AAA and CCC) whose amino acid is already known. By altering
the concentrations of the two nucleotides and analyzing the
polypeptides produced, the codons can be deduced.
◦ c. Copolymers with a known repeating sequence (e.g.,
UCUCUCUCU) will produce polypeptides with alternating
amino acids (e.g., leu-ser-leu-ser), indicating that UCU is one
and CUC is the other, but not which is which.
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It is a triplet code.
It is comma free. The mRNA is
read continuously, three bases at
a time, without skipping any
bases.
It is nonoverlapping. Each
nucleotide is part of only one
codon and is read only once
during translation.
It is almost universal, most
codons have the same amino
acid meaning. (Protozoan
Tetrahymena and mitochondria
of some organisms).
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It is degenerate. Of 20 amino acids, 18 are
encoded by more than one codon. Met (AUG)
and Trp (UGG) are the exceptions.
The code has start and stop signals. AUG is
the usual start defines the open reading
frame. Generally three stop codons: UAG
(amber), UAA (ochre), and UGA (opal).
Wobble occurs in the anticodon.
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Ribosomes translate the genetic message of
mRNA into proteins.
The mRNA is translated 5’ to 3’, producing a
corresponding N-terminal-to-C-terminal
polypeptide.
Amino acids are bound to tRNAs used by the
ribosome to build proteins.
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Amino acids are inserted into the polypeptide
in the proper sequence due to:
◦ a. Specific binding of each amino acid to its tRNA.
◦ b.Specific base-pairing between the mRNA codon
and tRNA anticodon.
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The tRNAs are 75–90 nt in length,
and each tRNA has a different
sequence.
All tRNAs can be shown in a
cloverleaf structure, with
complementary base pairing
between regions to form four
stems and loops. Loop II contains
the anticodon.
All tRNAs have CCA (added
posttranscriptionally) at their 3’
ends, for the amino attachement.
Extensive chemical modifications
are performed on all tRNAs after
transcription.
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Aminoacyl-tRNA synthetase attaches amino acids to their specific
tRNA molecules. The charging process (aminoacylation) produces a
charged tRNA (aminoacyl-tRNA), using energy from ATP hydrolysis.
There are 20 different
aminoacyl-tRNA synthetase
enzymes, one for each amino
acid, and each enzyme
recognizes the structure of the
specific tRNA(s) it charges.
The amino acid and ATP bind
to the specific aminoacyl-tRNA
synthetase enzyme.
The tRNA binds to the enzyme,
and the amino acid is
transferred onto it, displacing
the AMP. The aminoacyl-tRNA
is released from the enzyme.
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The amino acid is covalently
attached by its carboxyl
group to the 3’ end of the
tRNA. Every tRNA has a 3’
adenine, and the amino acid
is attached to the 3’-OH or
2’-OH of this nucleotide
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Ribosomes are the catalyst for protein
synthesis, facilitating binding of charged
tRNAs to the mRNA so that peptide bonds
can form.
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Ribosomes in both bacteria and eukaryotes consist of
two subunits of unequal size (large and small), each
with at least one rRNA and many ribosomal proteins
◦ a. Bacterial ribosomes are 70S, with 50S and 30S subunits.
 i. The 50S subunit contains the 23S rRNA and 5S rRNA.
 Ii. The 30S subunit contains the 16S rRNA.
◦ b. Mammalian ribosomes are 80S, with 60S and 40S subunits.
 i. The 60S subunit contains the 28S rRNA, the 5.8S rRNA, and the
5S.
 ii. The 40S subunit contains the 18S rRNA.
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When translating an mRNA,
specific sites on the ribosome
perform specific functions:
◦ a. The A site binds incoming
aminoacyl-tRNAs.
◦ b. The P site contains the tRNA
carrying the growing polypeptide
chain.
◦ c. The E site allows exit of the tRNA
after donating its amino acid.
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The P and A sites consist of
regions of both subunits of the
ribosome, while E is a region of
the large subunit.
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DNA regions that encode rRNA are called ribosomal DNA (rDNA)
or rRNA transcription units.
E. coli is a typical bacterium, with seven rRNA coding regions
scattered in its chromosome.
◦ a. Each rRNA transcription unit has a single promoter and contains the
genes 16S-23S-5S, in that order, with non-rRNA sequences as spacers.
◦ b. The pre-rRNA transcript associates with ribosomal proteins and is
cleaved by RNases to release the three rRNAs. These rRNAs associate with
ribosomal proteins to form functional ribosomal subunits.
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Eukaryotes generally have many copies of the rRNA genes.
◦ a. The three rRNA genes with homology to prokaryotic rRNA genes are
18S-5.8S-28S, in that order. In the chromosome these genes are tandemly
repeated 100–1,000 times to form rDNA repeat units.
◦ b. A nucleolus forms around each rDNA repeat unit and then they fuse to
make one nucleolus. Ribosomal subunits are produced in this structure by
addition of the 5S rRNA and ribosomal proteins.
◦ c. RNA polymerase I transcribes the rDNA repeat units, producing a prerRNA molecule containing the 18S, 5.8S, and 28S rRNAs, separated by
spacer sequences.
◦ d. The 5S rRNA gene copies are located elsewhere in the genome and are
transcribed by RNA polymerase III.
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Protein synthesis is similar in bacteria and
eukaryotes. Some significant differences will be
discussed.
In both, translation is divided into three stages:
◦ a. Initiation.
◦ b. Elongation.
◦ c. Termination.
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Initiation of translation requires:
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a. An mRNA.
b. A ribosome.
c. A specific initiator tRNA.
d. Initiation factors (IF).
e. GTP.
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Bacterial translation begins with
binding of the 30S ribosomal subunit
complexed with IF-1 and IF-3 to
mRNA near the AUG codon.
Ribosome binding to mRNA requires
more than the AUG:
◦ a. The ribosome binds at a ribosomebinding site (RBS), 8–12 nucleotides
upstream from the AUG, where it is
oriented to the correct reading frame for
protein synthesis.
◦ b. Discovered by Shine and Dalgarno,
these purine-rich sequences (AGGAG) are
complementary to the 3’ end of the 16S
rRNA.
◦ c. Further mutational analysis shows that
complementarity between the Shine–
Dalgarno sequence and the 3’ end of 16S
rRNA is important in ribosome binding to
the mRNA.
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Initiator tRNA binds the AUG to
which the 30S subunit is bound.
AUG universally encodes
methionine.
◦ a. Initiator methionine in bacteria is
formylmethionine (fMet). It is carried
by a specific tRNA (with the anticodon
5’-CAU-3’).
◦ b. The tRNA first binds a methionine,
and then transformylase attaches a
formyl group to the methionine,
making fMet-tRNA.fMET (a charged
initiator tRNA).
◦ c. Methionines at sites other than the
beginning of a polypeptide are
inserted by tRNA.Met (a different
tRNA), which is charged by the same
aminoacyl-tRNA synthetase as
tRNA.fMet.
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Met-tRNA.fMet is brought to the
30S-mRNA complex by IF-2/GTP.
IF-1 blocks the A site on the 30S
subunit, so only P site is open for
the tRNA.fMet, forming the 30S
initiation complex.
The 50S ribosomal subunit binds
the complex, GTP is hydrolyzed,
and the three IF are released,
forming a 70S initiation complex.
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Initiation of translation is similar in eukaryotes but more complex. Initiation
factors are called eIF. Main differences in eukaryotic translation:
a. Initiator methionine is not modified. As in prokaryotes, it is attached to a
special tRNA.
b. Ribosome binding involves the 5’ cap, not a
Shine–Dalgarno sequence.
◦ i.-Eukaryotic initiator factor (eIF-4F) is a protein
multimer, including cap-binding protein (CBP). It
binds the 5’ mRNA cap.
◦ ii.-Then the 40S subunit, complexed with initiator
Met-tRNA, several eIFs, and GTP, binds the cap
complex, including other eIFs.
◦ iii.-The initiator complex scans the mRNA for a Kozak
sequence including the (first) AUG start codon.
◦ iv.-Once there, 40S binds and then 60S binds,
displacing the eIFs and creating the 80S initiation
complex with initiator Met-tRNA in the ribosome’s P
site.
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c. The eukaryotic mRNA’s 3’ poly(A) tail also interacts with the 5’ cap. Poly(A)
binding protein (PABP) binds the poly(A) and binds a protein in eIF-4F on the
cap, circularizing the mRNA and stimulating translation.
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Elongation of the amino acid
chain has three steps:
◦ a. Binding of aminoacyl-tRNA to
the ribosome.
◦ b.Formation of a peptide bond.
◦ c. Translocation of the
ribosome to the next codon.
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Protein synthesis begins with
fMet-tRNA in the P site of the
ribosome. The next charged tRNA
approaches the ribosome bound
to EF-Tu-GTP. When the charged
tRNA hydrogen bonds with the
codon in the ribosome’s A site,
hydrolysis of GTP releases EFTu-GDP.
EF-Tu is recycled with assistance
from EF-Ts, which removes the
GDP and replaces it with GTP,
preparing EF-Tu-GTP to escort
another aminoacyl tRNA to the
ribosome.
The process is similar in
eukaryotes, with eEF-1A taking
the place of EF-Tu, and eEF-1B
the role of EF-Ts.
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The two aminoacyl-tRNAs are
positioned by the ribosome for
peptide bond formation, which
occurs in two steps:
◦ a. In the P site, the bond between the amino
acid and its tRNA is cleaved.
◦ b. Peptidyl transferase forms a peptide
bond between the now-free amino acid in
the P site and the amino acid attached to
the tRNA in the A site. 23S rRNA is most
likely the catalyst.
◦ c. The tRNA in the A site now has the
growing polypeptide chain attached to it.
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The ribosome now advances one codon along the mRNA. EF-G is used
in translocation in prokaryotes. EF-G-GTP binds the ribosome, GTP is
hydrolyzed, and the ribosome moves one codon while the uncharged
tRNA leaves the P site. In eukaryotes factor is called eEF-2.
Release of the uncharged tRNA involves the 50S ribosomal E (for Exit)
site. Binding to the A site is blocked until tRNA is released at the E site.
During translocation the peptidyl-tRNA remains attached to its codon
but is transferred from the ribosomal A site to the P site. This allows
release of the uncharged tRNA and EF-G, which will be reused.
The vacant A site contains a new codon, and an aminoacyl-tRNA with
the correct anticodon can enter and bind.
In both bacteria and eukaryotes, simultaneous translation occurs
creating a polyribosome (polysome) that efficiently produces many
polypeptides.
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Termination is signaled by
a stop codon (UAA, UAG,
UGA) that has no
corresponding tRNA.
Release factors (RF) assist
the ribosome in
recognizing the stop
codon and terminating
translation.
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In E. coli:
◦ i. RF1 recognizes UAA and UAG.
◦ ii. RF2 recognizes UAA and UGA.
◦ iii. RF3 stimulates termination via
GTP hydrolysis.
◦ Iv.-RRF (ribosome recycling factor)
binds the A site, EF-G translocates
the ribosome, RRF then releases
the last uncharged tRNA and EF-G
releases RRF, causing the
ribosomal subunits to dissociate
from the mRNA.
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In eukaryotes, eRF1 recognizes
all three stop codons, while
eRF3 stimulates termination.
Ribosome recycling occurs
without an equivalent of RRF
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Both bacteria and eukaryotes secrete proteins, and
eukaryotes must move proteins into intracellular
compartments. Signal sequences on the proteins direct
them to their destinations.
Blobel et al. (1975) showed that localization of the new
protein results from hydrophobic signal (leader)
sequences in the polypeptide.
In eukaryotes, proteins synthesized on the rough ER
(endoplasmic reticulum) are glycosylated and then
transported in vesicles to the Golgi apparatus. The
Golgi sorts proteins based on their signals and then
sends them to their destinations.
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The required signal sequence for a protein to enter the ER is 15–
30 N-terminal amino acids.
As the signal sequence is produced by translation, it is bound by
a signal recognition particle (SRP) composed of RNA and protein.
The SRP suspends translation until the complex binds a docking
protein on the ER membrane.
Once bound the signal sequence is inserted into the membrane,
SRP is released, and translation resumes. Synthesis through the
membrane into the ER, in an example of cotranslational transport.
In the ER cisternal space, the signal sequence is removed by
signal peptidase. The protein is usually glycosylated and then
transferred to the Golgi for sorting.