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L2: Protein Synthesis, Processing, and Regulation
Introduction
Outline
Translation is the synthesis of proteins as directed by
mRNA templates.
• Translation of mRNA
But it is only the first step in the formation of a
functional protein.
• Protein Folding and Processing
• Regulation of Protein Function
The polypeptide chain must then fold into the
appropriate conformation and often undergoes
various processing steps.
• Protein Degradation
Gene expression is regulated at the level of translation
in both prokaryotic and eukaryotic cells.
There are also multiple controls on the amounts and
activities of intracellular proteins, which ultimately
regulate all aspects of cell behavior.
Structure of tRNAs
tRNAs align amino acids with corresponding codons on the mRNA template.
They are 70 to 80 nucleotides long and have characteristic cloverleaf structures
resulting from complementary base pairing between different regions.
The anticodon loop binds to the appropriate codon by complementary base
pairing.
Translation of mRNA
Attachment of amino acids to specific tRNAs is mediated by
enzymes called aminoacyl tRNA synthetases.
Each of these 20 enzymes recognizes a single amino acid,
as well as the correct tRNA to attach it to.
The attachment occurs in two steps:
1. The amino acid is joined to AMP, forming aminoacyl
AMP.
2. The amino acid is transferred to the 3′ CCA terminus
of the tRNA and AMP is released.
The amino acid is then aligned on the mRNA template by
complementary base pairing.
At the third codon position, base pairing is relaxed, allowing
G to pair with U, and inosine (I) in the anticodon to pair
with U, C, or A.
The genetic code
Three different reading frames
Figure 6-50 Molecular Biology of the Cell (© Garland Science 2008)
Molecular Biology of the Cell (© Garland Science 2008)
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Nonstandard codon-anticodon base pairing (wobbling)
Nonstandard codon-anticodon base pairing
Molecular Biology of the Cell (© Garland Science 2008)
Attachment of amino acids to tRNAs
Translation of mRNA
Ribosomes are named according to sedimentation
rates in ultracentrifugation: 70S for bacterial and 80S
for eukaryotic.
Cells have many ribosomes, illustrating the
importance of protein synthesis. E. coli has about
20,000; growing mammalian cells can have ten
million.
All ribosomes have two subunits; each subunit
contains both rRNA and proteins.
The subunits of eukaryotic ribosomes are larger and
have more proteins than prokaryotic ribosomes.
Ribosome structure
Structure of 16S rRNA
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Translation of mRNA
Structure of the 50S ribosomal subunit
It was thought that ribosomal proteins catalyzed
protein synthesis, but later shown that rRNA has
catalytic activity.
Noller and colleagues in 1992 showed that the large
ribosomal subunit is able to catalyze formation of
peptide bonds (the peptidyl transferase reaction)
even after 90% of the ribosomal proteins have been
removed.
Unambiguous evidence for rRNA catalysis came from
high-resolution structural analysis of the 50S
ribosomal subunit, in 2000.
Ribosomal proteins are absent from the site of the
peptidyl transferase reaction showing that rRNA was
responsible for catalyzing peptide bond formation.
Translation of mRNA
Prokaryotic and eukaryotic mRNAs
It is now thought that ribosomal proteins play a largely
structural role, and the large ribosomal subunit
functions as a ribozyme.
This has evolutionary implications: RNAs are thought
to have been the first self-replicating
macromolecules.
The role of rRNA in the formation of peptide bonds
extends the catalytic activities of RNA beyond selfreplication to direct involvement in protein synthesis.
This may provide an important link for understanding
the early evolution of cells.
Translation of mRNA
Signals for translation initiation
In both prokaryotic and eukaryotic cells, translation always
initiates with methionine, usually encoded by AUG.
The signals that identify initiation codons are different in
prokaryotic and eukaryotic cells. Initiation codons in
bacterial mRNAs are preceded by a Shine-Dalgarno
sequence, that aligns the mRNA on the ribosome.
Thus they can initiate translation at the 5′ end of an mRNA
and at internal initiation sites of polycistronic mRNAs.
Eukaryotic mRNAs are recognized by the 7methylguanosine cap at the 5′ terminus.
The ribosomes then scan downstream until they encounter
the initiation codon.
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Overview of translation
3-5 aa/sec
100-200 aa in a min
30 000 aa (titin protein in muscels) in 2-3 h
Bacteria: ca. 15-20 aa/sec
Translation of mRNA
Initiation of translation in bacteria
In bacteria, initiation starts with a 30S ribosomal
subunit bound to initiation factors IF1 and IF3.
Then the mRNA, the initiator N-formylmethionyl
(fMet) tRNA, and IF2 (bound to GTP) join the
complex.
IF1 and IF3 are released, a 50S subunit binds to the
complex, and IF2 is released.
Initiation of translation in eukaryotic cells (Part 1)
Translation of mRNA
In eukaryotes initiation requires at least 11 proteins,
designated eIFs (eukaryotic initiation factors).
The initiator methionyl tRNA is bound to eIF2, and the
mRNA is brought to the complex by eIF4E.
The ribosome then scans down the mRNA to the first
AUG codon. This is accompanied by ATP hydrolysis.
Initiation factors are then released, and the 60S
subunit joins the complex.
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Initiation of translation in eukaryotic cells (Part 2)
Translation of mRNA
Initiation of translation in eukaryotic cells (Part 3)
Elongation stage of translation
The mechanism of elongation in prokaryotic and
eukaryotic cells is similar.
The ribosome has three binding sites: P (peptidyl), A
(aminoacyl), and E (exit) sites.
The initiator methionyl tRNA is bound at the P site.
The next aminoacyl tRNA binds to the A site by
pairing with the second codon of the mRNA.
An elongation factor (EF-Tu in prokaryotes, eEF1a in
eukaryotes) complexed to GTP brings the aminoacyl
tRNA to the complex.
Translation of mRNA
Selection of the correct aminoacyl tRNA determines the
accuracy of protein synthesis.
Translation of mRNA
Base pairing alone can’t account for the accuracy of protein
synthesis.
Then translocation occurs, during which the ribosome
moves three nucleotides along the mRNA,
positioning the next codon in an empty A site.
A “decoding center” in the small ribosomal subunit recognizes
correct codon-anticodon base pairs and discriminates
against mismatches.
This step translocates the peptidyl tRNA from A to P,
and the uncharged tRNA from P to E.
Insertion of a correct aminoacyl tRNA into the A site triggers a
conformational change that induces hydrolysis of GTP
bound to eEF1α and release of the elongation factor.
A new aminoacyl tRNA binds to the A site and induces
release of the uncharged tRNA from the E site.
Then the peptide bond is formed, catalyzed by the large
ribosomal subunit and the now uncharged initiator tRNA is at
the P site.
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Translation of mRNA
Regeneration of eEF1 α/GTP
As elongation continues, the eEF1α (or EF-Tu) released
from the ribosome bound to GDP must be reconverted to
its GTP form.
This requires a third elongation factor, eEF1βγ (EF-Ts in
prokaryotes).
Regulation of eEF1α by GTP binding and hydrolysis
represents a common method of protein regulation.
Elongation continues until a stop codon (UAA, UAG, or
UGA) is translocated into the A site.
Release factors recognize the signals and terminate
protein synthesis.
In prokaryotic cells RF1 recognizes UAA or UAG, RF2
recognizes UAA or UGA.
In eukaryotic cells eRF1 recognizes all three stop codons.
Termination of translation
Polysomes
Figure 6-76 Molecular Biology of the Cell (© Garland Science 2008)
Translation of mRNA
Translational regulation of ferritin
Translation of ferritin (a protein that stores iron) mRNA is regulated by repressor proteins.
When iron is absent, iron regulatory protein (IRP) binds to a the iron response element (IRE)
in the 5′ UTR, blocking translation.
Regulation of translation also plays a key role in
modulating gene expression.
Regulation includes translational repressor proteins and
noncoding microRNAs.
Global translational activity of cells is modulated in
response to cell stress, nutrient availability, and growth
factor stimulation.
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Translation of mRNA
Some translational repressors bind to specific sequences in
the 3′ UTR.
Some bind to initiation factor eIF4E, interfering with its
interaction with eIF4G and inhibiting initiation of translation.
Translation of mRNA
RNA interference (RNAi) mediated by short double-stranded
RNAs is used as an experimental tool to block gene expression
at the level of translation.
The RNA interference pathway inhibits translation and/or induces
degradation of target mRNAs.
Two types of small RNAs mediate RNA interference:
Small interfering RNAs (siRNAs)—produced from doublestranded RNAs by the nuclease Dicer.
MicroRNAs (miRNAs)—transcribed by RNA polymerase II, then
cleaved by nucleases Drosha and Dicer.
One strand is incorporated into the RNA-induced silencing
complex (RISC).
siRNAs or miRNAs that pair perfectly can induce cleavage of the
targeted mRNA.
Most miRNAs form mismatches that repress translation.
Regulation of translation by miRNAs (Part 1)
Regulation of translation by miRNAs (Part 2)
Translation of mRNA
Regulation of translation by phosphorylation of eIF2 and eIF2B
Translation can also be regulated by modification of initiation factors.
This results in global effects on overall translational activity rather than
translation of specific mRNAs.
Phosphorylation of eIF2 and eIF2B by regulatory protein kinases blocks
exchange of bound GDP for GTP, inhibiting initiation of translation.
Regulation of eIF4E: growth factors activate protein kinases that
phosphorylate regulatory proteins (eIF4E binding proteins, or 4E-BPs).
In the absence of growth factors, the nonphosphorylated 4E-BPs bind to
eIF4E and inhibit translation.
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Regulation of eIF4E
Protein Folding and Processing
Polypeptide chains must undergo
folding and other modifications to
become functional proteins.
The 3-D conformations result from
interactions between the side
chains of the amino acids.
All information for the correct
conformation is provided by the
amino acid sequence.
Molecular Biology of the Cell (© Garland Science 2008)
Current view of protein folding
Protein Folding and Processing
Chaperones are proteins that facilitate the folding of other
proteins.
They act as catalysts that facilitate assembly without becoming part
of the assembled complex.
They bind to and stabilize unfolded or partially folded polypeptides
that are intermediates leading to the final correctly folded state.
Chaperones bind to nascent polypeptide chains that are still being
translated on ribosomes.
The chain must be protected from aberrant folding or aggregation
with other proteins until synthesis of an entire domain is
complete.
Chaperones also stabilize unfolded polypeptide chains during their
transport into organelles.
Example: partially unfolded proteins stabilized by chaperones are
transported across the mitochondrial membrane.
Figure 6-85 Molecular Biology of the Cell (© Garland Science 2008)
Action of chaperones during translation
Chaperones in the mitochondrion then facilitate subsequent
folding.
Action of chaperones during protein transport
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Protein Folding and Processing
Sequential actions of chaperones
Many chaperones were initially identified as heat-shock
proteins (Hsp)—expressed in cells that were subjected to
high temperatures.
Hsp stabilize and facilitate refolding of proteins that have been
partially denatured.
Hsp70 chaperones and chaperonins are found in both
prokaryotic and eukaryotic cells.
Hsp70 proteins stabilize unfolded polypeptide chains during
translation by binding to short hydrophobic segments.
The unfolded polypeptide is then transferred to a chaperonin,
where folding takes place.
Chaperonins consist of protein subunits arranged in two
stacked rings to form a double-chambered structure. This
isolates the protein from the cytosol and other unfolded
proteins.
Hsp60-family (chaperonin) of molecular chaperones
Protein Folding and Processing
Two enzymes act as chaperones by catalyzing protein folding:
 Protein disulfide isomerase (PDI) catalyzes disulfide bond formation.
 PDI is one of the most abundant proteins in the ER, where an oxidizing
environment allows (S—S) linkages.
 Peptidyl prolyl isomerase catalyzes isomerization of peptide bonds that
involve proline residues.
 Isomerization between the cis and trans configurations of prolyl-peptide bonds,
could otherwise be a rate-limiting step in protein folding.
Figure 6-87 Molecular Biology of the Cell (© Garland Science 2008)
The role of signal sequences in membrane translocation
Protein Folding and Processing
Proteolysis—cleavage of the polypeptide chain removes
portions such as the initiator methionine from the amino
terminus.
Many proteins have amino-terminal signal sequences that
target the protein for transport to a specific destination.
The signal sequence is inserted into a membrane channel as it
emerges from the ribosome and the rest of the polypeptide
chain passes through as translation proceeds.
The signal sequence is then cleaved by a membrane protease
(signal peptidase).
Proteolytic processing also includes formation of active enzymes
or hormones by cleavage of larger precursors.
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Proteolytic processing of insulin
Protein Folding and Processing
Glycosylation adds carbohydrate chains to proteins
to form glycoproteins.
The carbohydrate moieties play important roles in
protein folding in the ER, in targeting proteins for
transport, and as recognition sites in cell-cell
interactions.
N-linked glycoproteins: the carbohydrate is attached
to the N atom in the side chain of asparagine.
O-linked glycoproteins: the carbohydrate is attached
to the O atom in the side chain of serine or
threonine.
Linkage of carbohydrate side chains to glycoproteins
Protein Folding and Processing
Some eukaryotic proteins are modified with lipids, which often serve to anchor them
to the plasma membrane.
There are four types of lipid additions:
1. N-myristoylation: myristic acid (a 14-carbon fatty acid) is attached to an N-terminal
glycine.
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These proteins are associated with the inner face of the plasma membrane.
2. Prenylation: prenyl groups are attached to sulfur atoms in the side chains of cysteine
located near the C terminus.

Many of these proteins are involved in control of cell growth and differentiation,
including the Ras oncogene proteins, which are responsible for many human
cancers.
3. Palmitoylation: palmitic acid (a 16-carbon fatty acid) is added to sulfur atoms of the
side chains of internal cysteine residues
4. Glycolipids (lipids linked to oligosaccharides) are added to C-terminal carboxyl groups.
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Addition of a fatty acid by N-myristoylation
They anchor the proteins to the external face of the plasma membrane.
The glycolipids contain phosphatidylinositol, and are called
glycosylphosphatidylinositol (GPI) anchors.
Prenylation of a C-terminal cysteine residue
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Palmitoylation
Structure of a GPI anchor
Feedback inhibition
Regulation of Protein Function
Regulation of protein function allows the cell to regulate not only the amounts
but also the activities of its proteins.
There are three general mechanisms of control of cellular proteins:
• regulation by small molecules
- most enzymes are controlled by changes in conformation, often as a result of
binding small molecules.
- end products of many biosynthetic pathways inhibit the enzymes that catalyze the
first step in their synthesis.
• phosphorylation
- phosphorylation is a reversible covalent modification process that can activate or
inhibit a wide variety of proteins in response to environmental signals.
- it is catalyzed by protein kinases, which transfer phosphate groups from ATP to
the hydroxyl groups of side chains of serine, threonine, or tyrosine.
- phosphorylation is reversed by protein phosphatases, which catalyze hydrolysis
of phosphorylated amino acids.
• protein-protein interactions
Protein kinases and phosphatases
Regulation of Protein Function
Other covalent modifications include methylation and
acetylation of lysine and arginine residues, and
nitrosylation—addition of NO groups to side chains
of cysteine.
O-linked glycosylation of proteins may also play a
regulatory role.
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Nitrosylation
Protein Degradation
Protein levels in cells are determined by rates of
synthesis and rates of degradation.
Differential rates of protein degradation are an
important aspect of cell regulation.
Many regulatory proteins have short half lives; this
allows levels to change quickly in response to
external stimuli.
Faulty or damaged proteins are recognized and
rapidly degraded.
The ubiquitin-proteasome pathway
Protein Degradation
The major pathway of protein degradation in eukaryotic cells is
the ubiquitin-proteasome pathway.
Ubiquitin is highly conserved in all eukaryotes; it is a marker
that targets proteins for rapid proteolysis.
Ubiquitin is attached to the amino group of the side chain of a
lysine residue, then more are added to form a chain.
Polyubiquinated proteins are recognized and degraded by a
large protease complex, the proteasome.
Ubiquitin can have other functions:
 Addition of one ubiquitin to some proteins is involved in
regulation of DNA repair, transcription, and endocytosis.
 SUMO (small ubiquitin-related modifier) and other ubiquitin-like
proteins serve as markers for protein localization and regulators
of protein activity.
Autophagy
Protein Degradation
Protein degradation can also take place in
lysosomes—membrane-enclosed organelles that contain
digestive enzymes, including proteases.
Lysosomes digest extracellular proteins taken up by endocytosis;
and take part in turnover of organelles and proteins.
Containment of digestive enzymes in lysosomes prevents
uncontrolled degradation of the cell contents.
Movement of proteins into lysosomes is accomplished by
autophagy: vesicles (autophagosomes) enclose small areas of
cytoplasm or organelles. The vesicles then fuse with lysosomes.
Autophagy is activated in nutrient starvation, allowing cells to degrade
nonessential proteins and organelles and reutilize the components.
Autophagy also plays a role in many developmental processes, such as
insect metamorphosis, which involve extensive tissue remodeling.
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Summary
• Translation is more complex in eukaryotes than in prokaryotes
• Translation can be regulated at the level of initiation and
elongation
- usually through control of the binding and hydrolysis of GTP
associated with initiation and elongation factors
- regulation often includes translational repressor proteins and
RNAi
• Translation has an inbuilt proof-reading mechanism
• Proteins undergo important processing steps/modifications:
- folding
- proteolysis (cleavage)
- glycosylation
- lipidification
- phosphorylation/dephosphorylation
- methylations/acetylations etc
that all have impact on the stability, activity, regulation and
function of the protein
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