CHAPTER 40
The Mechanism of Protein Synthesis
Problems: 2,3,6,7,9,13,14,15,18,19,20
• Initiation: Locating the start codon.
• Elongation: Reading the codons (5’→3’) and
synthesizing protein amino→carboxyl.
• Termination: Recognizing terminal codon
and releasing protein.
Prokaryotes
and Eukaryotes
40:1 Decoding the Information in
mRNA
Exit
Peptidyl
Aminoacyl
Initiation
Start codon (also GUG, Val)
Shine-Delgarno Sequence
Start:
1. Pairing of 16S
rRNA with SD of mRNA (a
UTR).
2. Pairing of
initiator tRNA
with start
codon.
Initiator tRNAf
Two different tRNA’s that
carry Met; tRNAf and tRNAm.
One Synthetase adds Met to
both tRNAf and tRNAm.
A specific transformylase only
formylates Met-tRNAf.
Initiation
Initiation factors, IF1, IF2
and IF3.
IF1 and IF3 prevent
premature assembly of the
70S ribosome.
IF2 binds to fMet-tRNAf
and mRNA at AUG codon.
The fMet-tRNAf in the fully
assembled ribosome is in the
P-site.
Initiation
Elongation Factor, EF-Tu
brings appropriate aa-tRNA
to the A-site.
fMet
AUG
Requires GTP: EF-Tu-GTP
Protects aa-tRNA ester
bond.
GTP→GDP only when proper
codon is present and then
EF-Tu is released.
40:2 Peptidyl Tranferase and Peptide
Bond Formation---Elongation
Peptidyl tranferase
center on 23S rRNA
of 50S subunit.
Catalysis occurs via
mechanism of
proximity and
orientation.
EF-Tu-GTP
EF-Ts
GDP
GTP
-EF-Tu-GTP
-EF-Tu-GDP
EF-Tu-GDP
Peptide grows from the H3N+-end to –COOH-end
Termination
Release Factors (RF) recognize the stop codons: RF1-UAA
or UAG and RF2-UAA or UGA.
RF recognizes stop codon and interacts with peptidyl
transferase promoting addition of H2O, rather than
aminoacyl-tRNA, to the growing peptide chain.
GTP hydrolysis after binding EF-G and binding of ribosome
release factor (RRF) cause release of ribosome.
40:3 Bacteria and Eukaryotes Differ
in Initiation of Protein Synthesis
Larger ribosome: 80S versus 70S.
Initiator tRNAi and Met-tRNAi is not
formylated; differs from Met-tRNAm.
Initiation involves eIF’s, CAP recognition
(eIF-4E), and movement to AUG start codon
(eIF-2).
Circular structure of eukaryotic mRNA
thought to facilitate rebinding of ribosomes
Important control
point. Mutation leads
to serious disease.
Elongation: EF1 and EF1 are counterparts
of EF-Tu and EF-Ts, and EF2 is equivalent of
bacterial EF-G translocase.
Termination: eRF1 is the release factor and is
the only release factor in eukaryotes. eIF-3
prevents premature ribosome reassembly in
absence of initiation factors.
Organization: In eukaryotes the translation
machinery is organized as a large complex with
the cytoskeleton.
40.4:
Antibiotic
Protein
Synthesis
inhibitors.
Diphtheria toxin
inhibits EF2 (the
eukaryotic
translocase).
40:5 Secretory and Membrane
Proteins
Protein targeting or sorting: 1. Posttranslational delivery to nucleus,
chloroplast, mitochondria, and peroxisomes. 2. Secretory pathway to
ER, and then the Golgi, lysosomes, integral membrane proteins, etc.
Signal
recognition
particle
Packaged in transport vesicles
and transported to location via
exo- and endocytosis
40:6 Protein Synthesis Regulation
Regulation by use of mRNA: Iron Response Elements (IRE)
Ferritin mRNA contains an IRE to
which an IRE binding protein (IRE-BP)
binds and blocks translation of the
mRNA.
High iron: IRE-BP binds iron and can
not bind ferritin mRNA. Ferritin is
produced to store iron.
Low iron: IRE-BP binds ferritin mRNA
and blocks ferritin production
Transferrin-receptor mRNA
contains an IRE to which an IRE
binding protein (IRE-BP) binds
under low iron that stabilizes the
mRNA, permitting translation and
production of the receptor.
Regulation Through Small RNAs
RNA interference, RNAi: External source of dsRNA is cleaved into
21-nucleotide fragments by an RNase → ss siRNA → RISC which
locats siRNA onto complementary mRNA and promotes their
degradation.
MicroRNAs, miRNA: miRNA produced from endogenous encoded
RNA precursor this binds to complementary region of mRNA forming
a 21-nucleotide ds RNA region which promotes degradation of that
mRNA. Human genome has more that 700 miRNAs, 60% of genes
regulated by one or more miRNAs: cell differentiation, development,
disease such as cancer, etc.