CHAPTER 30: PROTEIN SYNTHESIS (Translation)
Translation: mRNA
protein
LECTURE TOPICS
•
Complexity, stages, rate, accuracy
•
Amino acid activation [tRNA charging]
•
tRNAs and translating the Genetic Code
- Amino acid activation [tRNA charging]
- codon-anticodon recognition
- “Wobble” base pairing and code degeneracy
•
•
Ribosome Structure-mRNA/tRNA/rRNA base
pairings important in translation!
TRANSLATION PROCESS
- Prokaryotic
- Eukaryotic
- Inhibitors [specific steps in process]
Process Rates for Flow of Genetic Information (prokaryotic)
PROCESS
"MACHINERY"
RATES
DNA
REPLICATION
DNA
Polymerase I
10 bases/sec
DNA
Polymerase III
1000 bases/sec
RNA
Polymerase
50 bases/sec
TRANSCRIPTION
synthesis of mRNA for 200 a.a.
protein takes (200 x 3)/50 = 12
sec
TRANSLATION
Ribosomes
20 a.a.'s/sec
synthesis of a 200 a.a. protein
takes 200/20 =10 sec
Complexity of Translation: Cast of Characters
[amounts in one E.coli cell]
Translation machinery is 35% of dry weight of cell!
• 20,000 ribosomes
• 200,000 tRNAs
• 100,000 proteins and cofactors
Complexity of Translation: Stages and Cast of Characters
• tRNA is a compact A-helix structure!
• Only a few bases are NOT paired!
tRNA structure/animations: (Excellent!)
http://www3.interscience.wiley.com:8100/legacy/college/boyer/0471661791/structure/tRNA/trna.htm
Threonyl tRNAThr synthetase “Proofreading” (Error correcting)
Mistakes (Val, Ser): Actually
leaves Serine (1/100 a.a’s)
Hydrolyzes
wrong amino
acid (Ser)
a.a-AMP-Enzyme
Codon-anticodon recognition and “wobble” base pairing
3’
5’
UH2
UH2
Sometimes
inosine is at
5`-end of
anticodon
5’
I G C
mRNA codon
3’- C C
G
3’
-5’
“Wobble” I:C base pair
Wobble Hypothesis:
* Don’t need 61 tRNAs!
* The anticodon of some tRNAs can “read” 2 or 3 codons
(5`)
ONLY
(3`)
ONLY
Wobble basepairing
Ribosome Structure
30S
50S
70S
Transcription and translation are coupled in E.coli
They happen simultaneously!
Translation process is 100% Processive:
1. Make peptide bonds
2. Relative movements
(mRNA + ribosome)
3. Supply of reagents,etc.
Elongation
Termination
30S Initiation complex
Protein (polypeptide) Synthesis is NH2
COOH
Amino terminus
1
1
2
1
2
2
3
3
COOH terminus
Ribosome has two essential functions in protein synthesis
1
2
3’
5’
How does a ribosome work?
tRNA-mRNA-rRNA
base-pairing
interactions
determine
accuracy of
protein synthesis.
50S ribosome
subunit
Three
tRNAs
30S ribosome
subunit
mRNA
Purine-rich
Start
mRNAs
Initiation: Prokaryotic mRNA-16S rRNA base-pairing
Purine-rich
Start
rRNA-mRNA(codon)-tRNA(anticodon) base-pairing:
• another “accuracy” factor
Three tRNA binding sites on 30S subunit
CCA ends
Exit
Amino-acyl
Peptidyl
Codon-Anticodon
base pairing
[E,P,A] three tRNA binding sites on 30S subunit
NH2
[Large subunit]
3 tRNAs
[small subunit]
Protein Translation Factors
*
EF-Tu
23S rRNA
ribozyme
E P A
E P A
GTP
EF-G
GDP + Pi
Termination by
Release Factors
(Stop codon
recognition)
E P A
E P A
Elongation : Peptide Bond Synthesis
[peptide bond formed and growing peptide moves from P-site to A-site]
..
Initiator
tRNA
*
Catalyzed
by 23S
rRNA
ribozyme
50S Peptidyl transferase : A ribozyme activity
Adenine
2451
A site (Green)
P site(red)
P site
23S RNA-catalyzed peptide bond formation
A site
A site
Protein Synthesis Animation:
3-D model (Nierhaus, 2006)
Protein Synthesis Animation
Stop Dec 4, 2008
Eukaryotic cytoplasmic ribosome (80S)
Eukaryotic Initiation
complex
EIF-2(GTP) for start
AUG only
1) Ribosome binds to cap
2) Moves to 1st AUG
EIF2-GDP + Pi
3) Large + small subunits
associate
Elongation
Eukaryotic mRNA: Protecting the ends!
protein-RNA and protein-protein interactions circularize mRNA
eIF4E binds
to cap
eIF4E binds
to eIF4G
eIF4G binds
to PABI
Ricinus communis (castor bean) is the source of ricin, a
deadly poison that stops eukaryotic protein synthesis
(500μg is lethal human dose).
• A and B chains disulfide
linked
• B chain binds to cell
and induces uptake by
endocytosis
• A chain released in
cytoplasm
• is a N-glycosidase
that removes A4324 in
28S rRNA and
prevents elongation
factor binding