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

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

Ribosome structure

A

P P PPPPPP

P-sitepeptidyl tRNA site

A-siteaminoacyl tRNA site

mRNA5’

Small subunit

Large subunit

Ribosome with bound tRNAs and mRNA

mRNA5’ cap

40S subunit

M

eIF2

AUG

Initiator tRNA bound to thesmall ribosomal subunit with the

eukaryotic initiation factor-2 (eIF2)

Initiation of protein synthesis: mRNA binding

The small subunit finds the 5’ cap andscans down the mRNA to the first AUG codon

mRNA5’

40S subunit

M

eIF2

AUG

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

60S subunit

mRNA5’

M

AUG

• aminoacyl tRNA binds the A-site

• first peptide bond is formed

• initiation is complete

GCC

A

mRNA5’

M

AUG GCC

A

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

Cech (2000) Science 289:878-879Ban et al. (2000) Science 289:905-920Nissen et al. (2000) Science 289:920-930

Large ribosomal subunit

Protein (purple)lies on the surface

23S RNA (orange and white) makesup the core of the subunit

• Structure shows only RNAin the active site

• Adenine 2451 carries outacid-base catalysis

P

UCA

P

PPP

P

UCA GCA GGG UAG

A

P

PP

P

Elongation

GCA GGG UAG

• following peptide bond formationthe uncharged tRNA dissociatesfrom the P-site

• the ribosome shifts one codon alongthe mRNA, moving peptidyl tRNAfrom the A-site to the P-site; thistranslocation requires theelongation factor EF2

• the next aminoacyl tRNA thenbinds within the A-site; this tRNAbinding requires the elongationfactor EF1

• energy for elongation is provided bythe hydrolysis of two GTPs:• one for translocation• one for aminoacyl tRNA binding

EF1

EF2

P

UCA GCA GGG UAG

P

PP

P

Termination

• when translation reaches the stopcodon, a release factor (RF) bindswithin the A-site, recognizing thestop codon

• release factor catalyzes the hydrolysisof the completed polypeptide fromthe peptidyl tRNA, and the entirecomplex dissociates

RF

P

UCA GCA GGG UAG

PPPP

P

PP

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