8' Protein Synthesis and Protein Processing

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8. Protein Synthesis and Protein Processing
a). Ribosome structure b). Protein synthesis i).
Initiation of protein synthesis ii). Peptide
bond formation peptidyl transferase iii).
Elongation and termination iv). Inhibitors of
protein synthesis Antiviral action of
interferon Induction of 2-5A
synthase Induction of eIF2 kinase Antibiotics
c). Protein processing i). Synthesis of
secreted and integral membrane proteins ii).
Glycosylation and protein targeting iii).
Proteolytic processing
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• Learning Objectives for Lecture 8
• Understand the structure of the ribosome in the
  context of the translation process
• Understand the steps in the initiation of
  protein synthesis
• Understand the mechanism of peptide bond
  formation, and that it is RNA catalyzed
• Understand the processes of elongation and
  termination
• Understand how interferon inhibits viral protein
  synthesis
• Understand the mechanisms by which antibiotics
  inhibit protein synthesis and how some organisms
  become resistant to antibiotics
• Understand how secreted and membrane-bound
  proteins are synthesized
• Understand how proteins are glycosylated and
  what the functions of the carbohydrates are
• Understand the role of proteolytic processing in
  protein maturation

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Ribosome structure
P
P
P
P
Large subunit
P
P
P
A
P
P-site peptidyl tRNA site
A-site aminoacyl tRNA site
mRNA
5
Small subunit
Ribosome with bound tRNAs and mRNA
4
Initiation of protein synthesis mRNA binding
M
Initiator tRNA bound to the small ribosomal
subunit with the eukaryotic initiation factor-2
(eIF2)
eIF2
40S subunit
The small subunit finds the 5 cap and scans down
the mRNA to the first AUG codon
mRNA
5 cap
AUG
5
60S subunit

• the initiation codon is recognized
• eIF2 dissociates from the complex
• the large ribosomal subunit binds

eIF2
M
mRNA
5
AUG
40S subunit
6
A
M
mRNA
5
AUG
GCC
M
A

• aminoacyl tRNA binds the A-site
• first peptide bond is formed
• initiation is complete

mRNA
5
AUG
GCC
7
P-site
A-site

• Peptide bond formation
• peptide bond formation is
• catalyzed by peptidyl transferase
• peptidyl transferase is contained within
• a sequence of 23S rRNA in the
• prokaryotic large ribosomal subunit
• therefore, it is probably within
• the 28S rRNA in eukaryotes
• the energy for peptide bond formation
• comes from the ATP used in tRNA charging
• peptide bond formation results in a shift
• of the nascent peptide from the P-site

N
NH2 CH3-S-CH2-CH2-CH
OC O tRNA
NH2 CH3-CH OC O tRNA
C
NH2 CH3-S-CH2-CH2-CH
OC
OH tRNA
NH CH3-CH OC O tRNA
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Large ribosomal subunit
23S RNA (orange and white) makes up the core of
the subunit
Protein (purple) lies on the surface

• Structure shows only RNA
• in the active site
• Adenine 2451 carries out
• acid-base catalysis

Cech (2000) Science 289878-879 Ban et al. (2000)
Science 289905-920 Nissen et al. (2000) Science
289920-930
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Elongation
P

• following peptide bond formation
• the uncharged tRNA dissociates
• from the P-site

P
P
P
P

• the ribosome shifts one codon along
• the mRNA, moving peptidyl tRNA
• from the A-site to the P-site this
• translocation requires the
• elongation factor EF2

UCA
GCA GGG UAG
EF1
EF2

• the next aminoacyl tRNA then
• binds within the A-site this tRNA
• binding requires the elongation
• factor EF1

P
P
P
P
P

• energy for elongation is provided by
• the hydrolysis of two GTPs
• one for translocation
• one for aminoacyl tRNA binding

UCA
GCA GGG UAG
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Termination
RF
P
P

• when translation reaches the stop
• codon, a release factor (RF) binds
• within the A-site, recognizing the
• stop codon

P
P
P
UCA
GCA GGG UAG
P
P
P
P
P
P
P
P

• release factor catalyzes the hydrolysis
• of the completed polypeptide from
• the peptidyl tRNA, and the entire
• complex dissociates

UCA
GCA GGG UAG
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Induction and action of interferon
cell makes interferon in response to viral RNA
interferon binds to receptors on neighboring
cells and activates the cells
virus
cell cannot protect itself
virus invades cell
cell synthesizes antiviral proteins in response
to interferon activation
virus replicates
cell succumbs
virus invades neighboring cell
cell protected from viral infection by antiviral
proteins
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Functions of two antiviral proteins
inactive endonuclease
viral dsRNA
oligo 2-5 adenylate (2-5A)
ATP
2-5A synthase
-A-2-p-5-A-2-p-5A- N
active endonuclease viral mRNA degraded
interferon induces
P
eIF2 kinase
eIF2
eIF2
viral dsRNA
active
inactive viral protein synthesis cannot initiate
13
(No Transcript)
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Protein maturation modification, secretion,
targeting
3. the SRP docks with the SRP receptor on the
cytosolic side of the ER membrane and
positions the signal peptide for insertion
through a pore
Translation of a secreted protein
2. the signal recognition particlea (SRP)
binds the signal peptideb and halts
translation
ER lumen c cytosol
SRP
SRP receptor
5
AUG
polysome for secreted protein
1. translation initiates as usual on a
cytosolic mRNA
athe signal recognition particle (SRP) consists
of protein and RNA (7SL RNA) it binds to the
signal peptide, to the ribosome, and to the SRP
receptor on the ER membrane bthe signal peptide
is a polypeptide extension of 10-40 residues,
usually at the N-terminus of a protein, that
consists mostly of hydrophobic amino acids cER
endoplasmic reticulum
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4. translation resumes and the nascent
polypeptide moves into the ER lumen
5. signal peptidase, which is in the ER
lumen, cleaves off the signal peptide
ER lumen
cytosol
5
7. the ribosomes dock onto the ER membrane
the rough ER is ER studded with polysomes
6. the SRP is released and is recycled
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8. translation continues with the nascent
polypeptide emerging into the ER lumen
9. at termination of translation, the completed
protein is within the ER and is further
processed prior to secretion
completed protein is processed
and secreted
ER lumen
cytosol
5
UGA
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• Examples of secreted proteins
• polypeptide hormones (e.g., insulin)
• albumin
• collagen
• immunoglobulins

• Integral membrane proteins are also synthesized
  by the same mechanisms
• they may be considered partially secreted
• Examples of integral membrane proteins
• polypeptide hormone receptors (e.g., insulin
  receptor)
• transport proteins
• ion channels
• cytoskeletal anchoring proteins (e.g., band 3)

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• Glycosylation of proteins
• most integral membrane proteins and secreted
  proteins are glycosylated
• during translation on the ER membrane the
  protein begins to be glycosylated
• various oligosaccharide modifications occur in
  the ER and in the Golgi complex
• O-linked (Ser, Thr linked) oligosaccharides
  (linked to hydroxyl group)
• N-linked (Asn linked) oligosaccharides (linked
  to amide group)

Biosynthesis of N-linked oligosaccharides (first
7 steps)
Dolichol phosphate (polyprenol lipid carrier)
(2) UDP-
ER lumen
(1) UMP, (1) UDP
(5) GDP-
(5) GDP
reorientation
Cytosol
N-acetylglucosamine (GlcNAc) Mannose
Monosaccharides are transferred by specific
glycosyltransferases from nucleotide sugars
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Biosynthesis of N-linked oligosaccharides (second
7 steps)
ER lumen Dolicol-phosphates are the sugar donors
in the ER lumen they are synthesized in the
cytosol prior to being translocated to the lumen
(4)
Dolicol-P-mannose Dolicol-P-glucose
(3)
PP
Cytosol
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Transfer of oligosaccharide to protein
PP
Transfer of oligosaccharide chain to the growing
polypeptide
ER lumen
Asn I X I Ser (Thr)
Linkage is to the amide group of an
asparagine followed by any (X) amino acid (except
proline) followed by serine or threonine
Following synthesis, the protein is
transferred to the Golgi complex, where trimming
and further building of the oligosaccharides
occurs
Cytosol
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Formation of complex type oligosaccharides
Asn I X I Ser (Thr)
Trimming by glycosidases Building by
glycosyltransferases
common core structure
Asn I X I Ser (Thr)
Golgi lumen
A complex type oligosaccharide fucose
galactose sialic acid come from
nucleotide sugars translocated across the Golgi
membrane
Cytosol
The type of carbohydrate determines whether the
protein is targeted to the membrane, to a
vesicle, or is secreted
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Targeting of proteins to lysosomes (I-cell
disease)

• Proteins containing
• mannose-6-phosphate
• are targeted to lysosomes

Asn

• These proteins include the
• lysosomal hydrolases

UDP-

• Phosphate groups are added to
• mannose by transfer of GlcNAc
• phosphate from UDP-GlcNAc

Asn

• Patients with I-cell (for inclusion
• body) disease have a deficiency
• in the enzyme that transfers
• GlcNAc phosphate to mannose
• residues in the Golgi

P

• As a result, the hydrolases cannot
• be targeted to the lysosomes

Asn

• The resulting deficiency in
• lysosomal hydrolases results in
• an accumulation (inclusions)
• of material in the lysosomes

P
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Proteolytic processing
Processing of insulin (synthesized in the ER of
pancreatic b-cells)
Signal peptide
N
N
Disulfide bond formation
S I S
S I S
cleavage of signal peptide by signal peptidase
C
Proinsulin
C
Preproinsulin
B-chain
N
Insulin
S I S
S I S
Further trimming by a carboxypeptidase B-like
enzyme removes two basic residues
from each of the new ends
N
S I S
S I S
C
A-chain
N
C
C
C-chain
Cleavage by trypsin-like enzymes releases the
C-peptide
The C-chain is packaged in the
secretory vesicle and is secreted along with
active insulin
C-chain
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• Preproopiomelanocortin
• multiple functional polypeptides from a single
  precursor
• processed in a cell-specific manner

26aa 48aa 12aa 40aa
14aa 21aa 40aa 18aa
26aa
5aa
N
C
Signal peptide
Proopiomelanocortin
Corticotropin (ACTH)
g-MSH
b-Lipotropin
31aa
a-MSH
b-MSH
Endorphin
g-Lipotropin
Enkephalin (5aa)