Transcript Chapter 22 (Part 2)

Chapter 22 (Part 2)
Protein Synthesis
Translation
• Slow rate of synthesis (18 amino acids
per second)
• In bacteria translation and transcription
are coupled. As soon as 5’ end of mRNA
is synthesized translation begins.
• Situation in eukaryotes differs since
transcription and translation occur in
different cellular compartments.
Ribosomes
• Protein biosynthetic
machinery
• Made of 2 subunits
(bacterial 30S and 50S,
Eukaryotes 40S and 60S)
• Intact ribosome referred
to as 70S ribosome in
Prokaryotes and 80S
ribosome in Eukaryotes
• In bacteria, 20,000
ribosomes per cell, 20% of
cell's mass.
• Mass of ribosomes is
roughly 2/3 RNA
Prokaryotic Ribosome Structure
• E. coli ribosome is 25 nm
diameter, 2520 kD in mass,
and consists of two unequal
subunits that dissociate at
< 1mM Mg2+
• 30S subunit is 930 kD with
21 proteins and a 16S
rRNA
• 50S subunit is 1590 kD
with 31 proteins and two
rRNAs: 23S rRNA and 5S
rRNA
Eukaryotic Ribosome Structure
• Mitochondrial and chloroplast
ribosomes are quite similar to
prokaryotic ribosomes,
reflecting their supposed
prokaryotic origin
• Cytoplasmic ribosomes are
larger and more complex, but
many of the structural and
functional properties are similar
• 40S subunit contains 30
proteins and 18S RNA.
• 60S subunit contains 40
proteins and 3 rRNAs.
Ribosome Assembly
• Assembly is coupled w/ transcription
and pre-rRNA processing
Ribosome Structure
• Crystal structure of
ribosome is known
• mRNA is associated with
the 30S subunit
• Two tRNA binding sites (P
and A sites) are located in
the cavity formed by the
association of the 2
subunits.
• The growing peptide chain
threads through a “tunnel”
that passes through the
40S (30S in bacteria)
subunit.
Mechanics of Protein
Synthesis
• All protein synthesis involves three phases:
initiation, elongation, termination
• Initiation involves binding of mRNA and
initiator aminoacyl-tRNA to small subunit,
followed by binding of large subunit
• Elongation: synthesis of all peptide bonds with tRNAs bound to acceptor (A) and
peptidyl (P) sites.
• Termination occurs when "stop codon"
reached
Identification of Initiator Codon in
Prokaryotes
• Involves binding of initiator tRNA (Nformylmethionyl-tRNA) to initiator codon (first
AUG)
• The 30S subunit scans the mRNA for a specific
sequence (Shine-Dalgarno Sequence) which is
just upstream of the initiator codon. 16S RNA
is involved in recognition of S-D sequence.
Prokaryotic Translational Initiation
• Formation of Initiation
complex involves protein
initiation factors
• IF-3 keeps ribosome
subunits apart
• IF-2 identifies and binds
initiator tRNA. IF-2
must bind GTP to bind
tRNA.
• IF-1, IF-2, and IF-3
bind to 30S subunit to
form initiation complex
• Once 50S subunit binds
initiation complex, GTP is
hydrolyzed, initiator
tRNA enters P-site and
IFs disassociate
Eukaryotic Initiation of
Translation
• No S-D sequence.
• CAP binding protein (CBP) 5’ end of
mRNA by binding to 5’ CAP
structure
• An initiation complex forms with
CBP, initiation factors and the 40S
subunit.
• The complex then scans the mRNA
looking for the first AUG closest to
the 5’ end of the mRNA
• eIF-2 analogous to IF-2, transfers
tRNA to P sight. GTP hydrolysis
involed in release
Chain Elongation
Three step process:
1) Position correct aminoacyl-tRNA at
acceptor site
2) Formation of peptide bond between
peptidyl-tRNA at P site with
aminoacyl-tRNA at A site.
3) Shifting mRNA by one codon relative
to ribosome.
• Elongation Factor Tu
(EF-Tu) binds to
aminoacyl-tRNA and
delivers it to the A
site of the ribosome
• When EF-Tu binds
GTP a conformational
change occurs
allowing it to bind to
aminoacyl-tRNA.
• EF-Tu-tRNA
complex enters the
ribosome and
positions new tRNA
at A site.
• If the anticodon
matches the codon,
GTP is hydrolyzed
and EF-Tu releases
the tRNA and then
exits the ribosome.
Recycling of EF-Tu
• After leaving the
ribosome EF-TuGDP complex
associates with EFTscausing GDP to
disassociate.
• When GTP bind to
the EF-Tu/EF-Ts
complex, EF-Ts
disassociates and
EF-Tu can bind
another tRNA
Peptide Bond formation
P-Site
N
5' tRNA
N
O
H
H+
O
O
C
N
5' tRNA
H
OH
H
O
CH H
NH3+
N
N
N
O
H
5' tRNA
H
H
O
H
OH
N
N
H
H
OH
H
OH
5' tRNA
N
O
H
C
N
O
H
H
O
H
OH
C
CH H
H
BASE
NH2
N
N
O
O
H
A-Site
NH2
N
O
CH H
H
P-Site
NH2
N
N
O
O
H
A-Site
NH2
H
N
O
C
H
H
CH H
NH3+
N
N
Formation of
Peptide Bond
• Once the peptide bond
forms, the mRNA band
shifts to move the new
peptidyl-tRNA into the
P-site and moves the
deaminacyl-tRNA from
the E-site
• Binding of EF-GTP to
ribosome promotes the
translocation
• Hydrolysis of EF-GTP to
EF-GDP is required to
release EF from ribosome
and new cycle of
elongation could occur
More on elongation
• Growing peptide chain then
extends into the “tunnel” of
the 50S subunit.
• Floding of the native protein
does not occur until the
peptide exits the “tunnel”
• Folding is facilitated by
chaperones that are
associated with the ribosome
• To ensure the correct tRNA
enters the A site, the 16S
RNA is involved in determing
correct codon/anticodon
pairing at positions 1 and 2 of
the codon.
Eukaryotic elongation
process
• Similar to what occurs in prokaryotes.
• Analogous elongation factors.
• EF-1a = EF-Tu  docks tRNA in Asite
• EF-1b = EF-Ts  recycles EF-Tu
• EF-2 = EF-G  involved in
translocation process
Peptide Chain Termination
• Proteins known as "release factors" recognize
the stop codon (UGA, UAG, or UAA) at the A
site
• In E. coli RF-1 recognizes UAA and UAG, RF-2
recognizes UAA and UGA.
• RF-3 binds GTP and enhances activities of RF-1
and –2.
• Presence of release factors with a nonsense
codon at A site transforms the peptidyl
transferase into a hydrolase, which cleaves the
peptidyl chain from the tRNA carrier
• Hydrolysis of GTP is required for disassociation
of RFs, ribosome subunit and new peptide
Protein Synthesis is
Expensive!
• For each amino acid added to a
polypeptide chain, 1 ATP and 3
GTPs are hydrolyzed.
• This is the release of more energy
than is needed to form a peptide
bond.
• Most of the energy is need to
over-come entropy losses
Regulation of Gene Expression
RNA Processing
5’CAP
Active
enzyme
Post-translational
modification
mRNA
AAAAAA
RNA Degradation
Protein Degradation
Regulation of Protein
Synthesis
Regulation could occur at two levels in
translation
1) Initiation – formation of the
initiation complex
2) Elongation – elongation could be
stalled by if an mRNA contains
“rare” codons
Regulation of Globin
gene translation by
heme
• When heme is low,
HCI kinase
phosphorylates eIF-2GDP complex,
• GEF binds tightly to
phosphorylated eiF-2GDP complex
• prevents recycling of
eIF-2-GDP and stops
translation
Regulation of the trp operon
• Transcription and translation are
tightly coupled in E. coli.
• When Trp is aundant, transcription of
the trp operon is repressed.
• The mechanism of this repression is
related to translation of the