8. Protein Synthesis and Protein Processing
a). Ribosome structureb). Protein synthesis
i). Initiation of protein synthesisii). Peptide bond formation; peptidyl transferaseiii). Elongation and terminationiv). Inhibitors of protein synthesis
Antiviral action of interferonInduction of 2-5A synthaseInduction of eIF2 kinase
Antibioticsc). Protein processing
i). Synthesis of secreted and integral membrane proteinsii). Glycosylation and protein targetingiii). Proteolytic processing
C
NH2
CH3-S-CH2-CH2-CH O=C
Peptide bond formation
• peptide bond formation iscatalyzed by peptidyl transferase
• peptidyl transferase is contained withina sequence of 23S rRNA in theprokaryotic large ribosomal subunit;therefore, it is probably withinthe 28S rRNA in eukaryotes
• the energy for peptide bond formationcomes from the ATP used in tRNA charging
• peptide bond formation results in a shiftof the nascent peptide from the P-siteto the A-site
NH2
CH3-S-CH2-CH2-CH O=C O
tRNA
NH2
CH3-CH O=C O
tRNA
N
P-site A-site
OH
tRNA
NHCH3-CH O=C O
tRNA
Induction and action of interferon
virus
virus invades cell
cell makes interferonin response to viral RNA
cell cannotprotect itself
virus replicates
cell succumbs
interferon binds toreceptors on neighboring cells
and activates the cells
cell synthesizesantiviral proteins
in response tointerferon activation
virus invades neighboring cell
cell protected from viralinfection by antiviral proteins
Functions of two antiviral proteins
interferoninduces
ATPviral dsRNA
2-5A synthaseoligo 2-5 adenylate (2-5A)
[-A-2’-p-5’-A-2’-p-5’A-] N
eIF2viral dsRNA
eIF2 kinaseeIF2
P
active inactive:viral protein synthesis cannot initiate
inactiveendonuclease
activeendonuclease:
viral mRNA degraded
Inhibitors of protein synthesis
Inhibitor Process Affected Site of Action Kasugamycin initiator tRNA binding 30S subunitStreptomycin initiation, elongation 30S subunitTetracycline aminoacyl tRNA binding A-siteErythromycin peptidyl transferase 50S subunitLincomycin peptidyl transferase 50S subunitClindamycin peptidyl transferase 50S subunit Chloramphenicol peptidyl transferase 50S subunit
Staphylococcus resistance to erythromycin
• certain strains of Staphylococcus can carry a plasmid that encodesan RNA methylase
• this RNA methylase converts a single adenosine residue in 23S rRNAto N6-dimethyladenosine
• this is the site of action of erythromycin, lincomycin, and clindamycin• N6-dimethyladenosine blocks the action of these antibiotics• the organism that produces erythromycin has its own RNA methylase
and thus is resistent to the antibiotic it makes
Protein maturation: modification, secretion, targeting
5’ AUG
polysome for secreted protein
2. the signal recognition particlea (SRP)
binds the signal peptideb and halts translation
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 membranebthe signal peptide is a polypeptide extension of 10-40 residues, usually at the N-terminus of a protein, that consists mostly of hydrophobic amino acidscER = endoplasmic reticulum
ER lumen c
cytosol
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
SRP SRP receptor
Translation of a secreted protein
5’
ER lumen
cytosol
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
7. the ribosomes dock onto the ER membrane; the rough ER is ER studded with polysomes
6. the SRP is released and is recycled
5’
ER lumen
cytosol
UGA
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 andsecreted
• 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)
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)
ER lumen
Cytosol
P
(1) UMP, (1) UDP
Dolichol phosphate (polyprenol lipid carrier)
N-acetylglucosamine (GlcNAc) =
Mannose =
(2) UDP-
PP
(5) GDP-
(5) GDP
PP
reorientation
Monosaccharides are transferredby specific glycosyltransferases
from nucleotide sugars
PP ER lumen
Dolicol-phosphates are thesugar donors in the ER lumen;
they are synthesized in the cytosolprior to being translocated to the lumen
Cytosol
PP
PP
(4)
(3)Dolicol-P-mannose =
Dolicol-P-glucose =
P
P
PP
Biosynthesis of N-linked oligosaccharides (second 7 steps)
ER lumen
Cytosol
PP
Linkage is to the amide group of an asparaginefollowed by any (X) amino acid (except proline)
followed by serine or threonine
Transfer of oligosaccharide chainto the growing polypeptide
AsnIXI
Ser (Thr)
Following synthesis, the protein is transferredto the Golgi complex, where trimming and further
building of the oligosaccharides occurs
Transfer of oligosaccharide to protein
AsnI
XI
Ser (Thr)
AsnI
XI
Ser (Thr)
Trimming by glycosidases;Building by glycosyltransferases
A complex type oligosaccharide
fucose = galactose = sialic acid =come from nucleotide sugars translocated
across the Golgi membrane
Golgi lumen
CytosolThe type of carbohydrate determines whether
the protein is targeted to the membrane,to a vesicle, or is secreted
= common core structure
Formation of complex type oligosaccharides
Targeting of proteins to lysosomes (I-cell disease)
Asn
Asn
UDP-
P
P
Asn
P
P
• Proteins containing mannose-6-phosphate are targeted to lysosomes
• Patients with I-cell (for inclusion body) disease have a deficiency in the enzyme that transfers GlcNAc phosphate to mannose residues in the Golgi
• Phosphate groups are added to mannose by transfer of GlcNAc phosphate from UDP-GlcNAc
• The resulting deficiency in lysosomal hydrolases results in an accumulation (inclusions) of material in the lysosomes
• These proteins include the lysosomal hydrolases
• As a result, the hydrolases cannot be targeted to the lysosomes
Proteolytic processing
Processing of insulin (synthesized in the ER of pancreatic -cells)
N
C
Preproinsulin
cleavage ofsignal peptideby signalpeptidase
Signal peptide
C
SI
S
SI
S
N
Proinsulin
C
SI
S
SI
S
N
C-chain
Cleavage by trypsin-like enzymesreleases the C-peptide
C
SI
S
SI
S
N
Insulin
CN
Disulfide bondformation
Further trimming by a carboxypeptidase B-like enzyme removes two basic residues from each of the new ends
C-chain The C-chain is packaged in the secretoryvesicle and is secreted along with active insulin
B-chain
A-chain
Preproopiomelanocortin• multiple functional polypeptides from a single precursor• processed in a cell-specific manner
26aa 48aa 12aa 40aa 14aa 21aa 40aa 18aa 26aaN C
Signalpeptide
Proopiomelanocortin
Corticotropin(ACTH)
-MSH -Lipotropin
-MSH -MSH Endorphin
-Lipotropin Enkephalin (5aa)
31aa
5aa
8. Protein Synthesis and Protein Processing
a). Ribosome structureb). Protein synthesis
i). Initiation of protein synthesisii). Peptide bond formation; peptidyl transferaseiii). Elongation and terminationiv). Inhibitors of protein synthesis
Antiviral action of interferonInduction of 2-5A synthaseInduction of eIF2 kinase
Antibioticsc). Protein processing
i). Synthesis of secreted and integral membrane proteinsii). Glycosylation and protein targetingiii). Proteolytic processing
C
NH2
CH3-S-CH2-CH2-CH O=C
Peptide bond formation
• peptide bond formation iscatalyzed by peptidyl transferase
• peptidyl transferase is contained withina sequence of 23S rRNA in theprokaryotic large ribosomal subunit;therefore, it is probably withinthe 28S rRNA in eukaryotes
• the energy for peptide bond formationcomes from the ATP used in tRNA charging
• peptide bond formation results in a shiftof the nascent peptide from the P-siteto the A-site
NH2
CH3-S-CH2-CH2-CH O=C O
tRNA
NH2
CH3-CH O=C O
tRNA
N
P-site A-site
OH
tRNA
NHCH3-CH O=C O
tRNA
Induction and action of interferon
virus
virus invades cell
cell makes interferonin response to viral RNA
cell cannotprotect itself
virus replicates
cell succumbs
interferon binds toreceptors on neighboring cells
and activates the cells
cell synthesizesantiviral proteins
in response tointerferon activation
virus invades neighboring cell
cell protected from viralinfection by antiviral proteins
Functions of two antiviral proteins
interferoninduces
ATPviral dsRNA
2-5A synthaseoligo 2-5 adenylate (2-5A)
[-A-2’-p-5’-A-2’-p-5’A-] N
eIF2viral dsRNA
eIF2 kinaseeIF2
P
active inactive:viral protein synthesis cannot initiate
inactiveendonuclease
activeendonuclease:
viral mRNA degraded
Inhibitors of protein synthesis
Inhibitor Process Affected Site of Action Kasugamycin initiator tRNA binding 30S subunitStreptomycin initiation, elongation 30S subunitTetracycline aminoacyl tRNA binding A-siteErythromycin peptidyl transferase 50S subunitLincomycin peptidyl transferase 50S subunitClindamycin peptidyl transferase 50S subunit Chloramphenicol peptidyl transferase 50S subunit
Staphylococcus resistance to erythromycin
• certain strains of Staphylococcus can carry a plasmid that encodesan RNA methylase
• this RNA methylase converts a single adenosine residue in 23S rRNAto N6-dimethyladenosine
• this is the site of action of erythromycin, lincomycin, and clindamycin• N6-dimethyladenosine blocks the action of these antibiotics• the organism that produces erythromycin has its own RNA methylase
and thus is resistent to the antibiotic it makes
Protein maturation: modification, secretion, targeting
5’ AUG
polysome for secreted protein
2. the signal recognition particlea (SRP)
binds the signal peptideb and halts translation
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 membranebthe signal peptide is a polypeptide extension of 10-40 residues, usually at the N-terminus of a protein, that consists mostly of hydrophobic amino acidscER = endoplasmic reticulum
ER lumen c
cytosol
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
SRP SRP receptor
Translation of a secreted protein
5’
ER lumen
cytosol
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
7. the ribosomes dock onto the ER membrane; the rough ER is ER studded with polysomes
6. the SRP is released and is recycled
5’
ER lumen
cytosol
UGA
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 andsecreted
• 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)
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)
ER lumen
Cytosol
P
(1) UMP, (1) UDP
Dolichol phosphate (polyprenol lipid carrier)
N-acetylglucosamine (GlcNAc) =
Mannose =
(2) UDP-
PP
(5) GDP-
(5) GDP
PP
reorientation
Monosaccharides are transferredby specific glycosyltransferases
from nucleotide sugars
PP ER lumen
Dolicol-phosphates are thesugar donors in the ER lumen;
they are synthesized in the cytosolprior to being translocated to the lumen
Cytosol
PP
PP
(4)
(3)Dolicol-P-mannose =
Dolicol-P-glucose =
P
P
PP
Biosynthesis of N-linked oligosaccharides (second 7 steps)
ER lumen
Cytosol
PP
Linkage is to the amide group of an asparaginefollowed by any (X) amino acid (except proline)
followed by serine or threonine
Transfer of oligosaccharide chainto the growing polypeptide
AsnIXI
Ser (Thr)
Following synthesis, the protein is transferredto the Golgi complex, where trimming and further
building of the oligosaccharides occurs
Transfer of oligosaccharide to protein
AsnI
XI
Ser (Thr)
AsnI
XI
Ser (Thr)
Trimming by glycosidases;Building by glycosyltransferases
A complex type oligosaccharide
fucose = galactose = sialic acid =come from nucleotide sugars translocated
across the Golgi membrane
Golgi lumen
CytosolThe type of carbohydrate determines whether
the protein is targeted to the membrane,to a vesicle, or is secreted
= common core structure
Formation of complex type oligosaccharides
Targeting of proteins to lysosomes (I-cell disease)
Asn
Asn
UDP-
P
P
Asn
P
P
• Proteins containing mannose-6-phosphate are targeted to lysosomes
• Patients with I-cell (for inclusion body) disease have a deficiency in the enzyme that transfers GlcNAc phosphate to mannose residues in the Golgi
• Phosphate groups are added to mannose by transfer of GlcNAc phosphate from UDP-GlcNAc
• The resulting deficiency in lysosomal hydrolases results in an accumulation (inclusions) of material in the lysosomes
• These proteins include the lysosomal hydrolases
• As a result, the hydrolases cannot be targeted to the lysosomes
Proteolytic processing
Processing of insulin (synthesized in the ER of pancreatic -cells)
N
C
Preproinsulin
cleavage ofsignal peptideby signalpeptidase
Signal peptide
C
SI
S
SI
S
N
Proinsulin
C
SI
S
SI
S
N
C-chain
Cleavage by trypsin-like enzymesreleases the C-peptide
C
SI
S
SI
S
N
Insulin
CN
Disulfide bondformation
Further trimming by a carboxypeptidase B-like enzyme removes two basic residues from each of the new ends
C-chain The C-chain is packaged in the secretoryvesicle and is secreted along with active insulin
B-chain
A-chain
Preproopiomelanocortin• multiple functional polypeptides from a single precursor• processed in a cell-specific manner
26aa 48aa 12aa 40aa 14aa 21aa 40aa 18aa 26aaN C
Signalpeptide
Proopiomelanocortin
Corticotropin(ACTH)
-MSH -Lipotropin
-MSH -MSH Endorphin
-Lipotropin Enkephalin (5aa)
31aa
5aa