Protein Biosynthesis II

• mRNA BindingShine-Dalgarno Sequence

• Initiation Complexf-Met-tRNAf

Met

Initiation factors

• ElongationElongation factors

• TerminationTermination factors

• mRNA BindingShine-Dalgarno Sequence

• Initiation Complexf-Met-tRNAf

Met

Initiation factors

• ElongationElongation factors

• TerminationTermination factors

Dintzis Experiment

COO-

5’

Trypsin

H2N-

H2N-COO-

C-terminal is richerin radioactive 3H-leucine

C-terminal is richerin radioactive 3H-leucine

3H-Leucine

H2N-

H2N-

3’

Released Protein4 min

8 min

60 min

3H

C-terminalN

Product analysis

Shine-Dalgarno

Purine-rich mRNA

Pyrimidine-rich rRNA complementary

fMet-tRNAfMet

CH3-S-CH2CH2C-COO-tRNA

H

N-H

C=OH

N-Terminal Block

Blocking the N-terminal amino acid assures thatthe peptide chain will grow towards the C-terminal

Blocking the N-terminal amino acid assures thatthe peptide chain will grow towards the C-terminal

Vocabulary of Terms• Aminoacyl-tRNA: tRNA with an amino acid

attached• Peptidyl-tRNA: tRNA with peptide attached• Nascent chain: peptide chain in act of being

assembled• P-site: Site on ribosome where peptidyl-tRNA sits• A-site: Site on ribosome where incoming

aminoacyl-tRNA binds• Peptidyl transferase: Enzyme that forms peptide

bond

Protein Biosynthesis takes place on Ribosomes

Picture the ribosomes as an assembly site and a ratchet machine

GTP, not ATP, is the fuel that drives the machine

tRNAS are brought to the assemble site called the “A” site

A peptide bond is formed by transferring the peptide from the P site to the N-terminal of the new amino acid (aminoacyl-tRNA)

OVERALL MECHANISMOVERALL MECHANISM

The peptide now has the new amino acid in the C-terminal…still attached to the tRNA

The peptidyl tRNA is in the A site

The mRNA with the peptidyl-tRNA attached shifts one frame bringing a newcodon into the A site

Simultaneously, the peptidyl tRNA is pushedinto the P site and the uncharged tRNA isshoved into the E site and expelled

(OVERALL CONT.)(OVERALL CONT.)

1. The machine is disassembled into its component parts to allow the mRNA to bind

2. The starting tRNA is set into position

3. The larger 50S subunit is put back inplace

INITIATIONINITIATION

Initiation Complex

ElongationElongation

2. An aminoacyl-tRNA reacts with elongationfactor EF-Tu in preparation for bindingto the A site.

1. Peptidyl-tRNA sits in the P site

3. A-site binding occurs as GTP is hydrolyzed.EF-Ts removes spent GDP.

4. A peptide bond is formed with the transferof the peptide from the P site to the A site

5. Elongation factor EF-G shifts the mRNAone frame, expelling the uncharged tRNA

Elongation

TerminationTermination

2. A termination factor that recognizes the codon binds to the A site

3. The peptide in the P site is transferredto the factor

1. Translocation results in a stopcodon (UAA, UGA, UAG) in the A site

4. Because the factor cannot bind,the peptide is released

Termination COOH

Diphtheria Toxin (Cornyebacterium diphtheria with

1. Reacts with elongation factor eEF-2 (prokaryote G)

eEF-2 + NAD+

bacteriophage cornyephage )

N

CONH2

O

OH

CH2OO-P-O-P-

OO

OOO CH2

N

N

N

N

NH2

OHHO HO

+

Break glycosidic bond to nicotinamide in reaction

ss

Trypsin

ss

A (catalytically active)

B

Diphtheria toxin

(cell penetration)NH2

N

N

CH2CH2 CH

C=O

N(CH3)3+

PEPTIDE CHAIN

NH2

N

N

CH2CH2 CH

C=O

N(CH3)3+

PEPTIDE CHAIN

O

OHHO

CH2ADPNAD+

eEf-2(diphthamide group)

Modified His in eEF-2 is target

ADP ribosylation

ss

A

B

Receptor

ss

A

BsH

Endocytosis

Disulfide cleavage

s

A

NAD+

eEF-2

ADP-ribosyl-eEF-2

Active enzyme

H

(ribosylates histidineresidues with diphthamide structure)

(See p 879)

Translation Blocking Antibiotics

Puromycin

Binds to A site andreceives peptidefrom transpeptidase

Aborts peptideprematurely

Prokaryotes and eukaryotes

Aminoglycoside

Causes misreading and inhibits chain initiation of prokaryote mRNA

Blocks peptidyl transferase inprokaryotes (A-site binding)

Inhibits aminoacyl-tRNA bindingto prokaryotes: blocks nutritionalfactor ppGpp (p867)

Post-translational Processing

• Folding

• Proteolytic processing

• Additions to peptide chainCarbohydrates (glycosylation)Methyl groups Lipid groups

Hydroxylation, deamination

• Secretion

FoldingRule: Proteins must fold to an active conformation. Generally, folding is spontaneous as the protein seeks its lowest energy level

Rule: Cells have “chaperones” that assist in proper folding. Typical are Hsp70 and Hsp60

Random ChainRandom Chain

Secondary helix or sheet

Secondary helix or sheet

Hydrophobic alignments

Hydrophobic alignments

Compact folded protein

Compact folded protein

StepwiseStepwise

Proteolytic Processing of Insulin

A chain

Preproinsulin Proinsulin Insulin

B chainDisulfide bonds

sss

ss s

s s