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
Antibiotics
Learning Objectives:
• 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 the mechanisms by which antibiotics inhibit
protein synthesis and how some organisms become
resistant to antibiotics
 Ribosome structure

P PP
P
P Large subunit
P
P
P A
P-site A-site
peptidyl tRNA site aminoacyl tRNA site

5’ mRNA

Small subunit

Ribosome with bound tRNAs and mRNA

 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

5’ cap AUG mRNA

 60S subunit
• the initiation codon is recognized
• eIF2 dissociates from the complex
• the large ribosomal subunit binds

eIF2

5’ AUG mRNA

40S subunit
 A
M

5’ AUG GCC mRNA

M
A
• aminoacyl tRNA binds the A-site

• first peptide bond is formed

5’ AUG GCC mRNA

• initiation is complete
Peptide bond formation P-site A-site
NH2 N
NH2
CH3-S-CH2-CH2-CH CH3-CH
C
O=C O=C
O O

tRNA tRNA
• peptide bond formation is
catalyzed by peptidyl transferase
• peptidyl transferase is contained within
a sequence of 23S rRNA in the NH2
prokaryotic large ribosomal subunit; CH3-S-CH2-CH2-CH
therefore, it is probably within
the 28S rRNA in eukaryotes
O=C
• the energy for peptide bond formation
NH
comes from the ATP used in tRNA charging CH3-CH
• peptide bond formation results in a shift O=C
of the nascent peptide from the P-site OH O
to the A-site
tRNA tRNA
Elongation
P
P • following peptide bond formation
P the uncharged tRNA dissociates
P
P from the P-site

• the ribosome shifts one codon along

UCA GCA GGG UAG the mRNA, moving peptidyl tRNA
from the A-site to the P-site; this
translocation requires the
EF1 elongation factor EF2

EF2 A
• the next aminoacyl tRNA then
P binds within the A-site; this tRNA
P
P binding requires the elongation
P factor EF1
P

• energy for elongation is provided by

UCA GCA GGG UAG the hydrolysis of two GTPs:
• one for translocation
• one for aminoacyl tRNA binding
Termination
RF
P
P
P • when translation reaches the stop
P
P codon, a release factor (RF) binds
within the A-site, recognizing the
stop codon
UCA GCA GGG UAG

PPPP
PP
P
P
• release factor catalyzes the hydrolysis
of the completed polypeptide from
the peptidyl tRNA, and the entire
complex dissociates
UCA GCA GGG UAG
 Inhibitors of protein synthesis

Inhibitor Process Affected Site of Action

Kasugamycin initiator tRNA binding 30S subunit
Streptomycin initiation, elongation 30S subunit
Tetracycline aminoacyl tRNA binding A-site
Erythromycin peptidyl transferase 50S subunit
Lincomycin peptidyl transferase 50S subunit
Clindamycin peptidyl transferase 50S subunit
Chloramphenicol peptidyl transferase 50S subunit

Staphylococcus resistance to erythromycin

• certain strains of Staphylococcus can carry a plasmid that encodes

an RNA methylase
• this RNA methylase converts a single adenosine residue in 23S rRNA
to 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