Translation: RNA-Directed Polypeptide Synthesis
Translation: Like making a Honda
one part at a time
Translation: Machinery and Process
are Complex
Protein Synthesis Animation:
3-D model (Nierhaus, 2006)
Protein Synthesis Animation
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
•
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
How does a ribosome work?
NH2
[Large subunit]
tRNAs
[small subunit]
tRNA Common Features
*
*
*
*
*
* Modified
bases can
prevent charging
with a wrong
amino acid
*
*
Major tRNA Structure Features
*
*
•
•
•
•
Compact
L- Shaped
Mostly base-paired A-helix
Unusual base pairs (3 bases)
*
Bases
face out
for pairing
*
• tRNA is a compact A-helix structure!
• Only a few bases are NOT paired!
*tRNA Movie*
Protein (polypeptide) Synthesis is NH2
COOH
Amino terminus
1
2
2
1
3
2
3
1
COOH terminus
2 Classes of aminoacyl-tRNA Synthetases:
[Recognize 2 different conformations of CCA]
How can an aminoacyl-tRNA synthetase
discriminate between similar amino acids??
Ex: Threonine tRNAThr amino acid charging (“Activation”)
[Discriminate between Thr, Val, Ser]
Threonyl-tRNAThr
synthetase
(CCA)
tRNAThr
1st step is formation of a.a.-AMP-enzyme Intermediate:
[activation is an ATP requiring reaction with PPi as a product]
Threonyl tRNAThr synthetase “Proofreading” (Error correcting)
Mistakes: Actually leaves
Serine (1/100 a.a’s)
Hydrolyzes
wrong amino
acid (Ser)
a.a-AMP-Enzyme
tRNA - mRNA recognition determined by?
[YES] codon-anticodon base-pairing
[NO] what amino acid is on the tRNA
If Ala-tRNACys used:
get alanine where
cysteine should be
CHAPTER 30: PROTEIN SYNTHESIS (Translation)
Translation: mRNA
protein
LECTURE TOPICS
•
•
•
tRNAs and translating the Genetic Code
- “Wobble” base pairing and code
degeneracy
Ribosome Structure
TRANSLATION PROCESS
- Prokaryotic
- Eukaryotic
- Inhibitors [specific steps in process]
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
C
I
A
Inosine
“wobble”
Base pairs
I
U
I
“Wobble” codon base (3’) pairs with (5`) - I of anticodon
[3 Ala codons read by only one tRNA anticodon]
The E. coli ribosome
Small subunit
Large subunit
Subunits
associated for
protein
synthesis
16S rRNA Structure
(1542 bases long)
5` end
secondary
structure
3` end
3-D X- ray
structure
Ribosome Structure
30S
50S
70S
30S Facing Solvent (outer surface)
More protein-rich than other side [next slide]
30S Facing 50S
[*30S Movie*]
Mostly RNA at this surface
50S
ribosome
subunit
Facing 30S
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
Ribosome has two essential functions in protein synthesis
1
1
2
2
3’
5’
tRNA-mRNA-rRNA base-pairing interactions
determine accuracy of protein synthesis.
50S ribosome
subunit
Three
tRNAs
30S ribosome
subunit
mRNA
50S Ribosome subunit: Protein Exit Channel
Hydrophobic channel is 100 Angstrom diameter
Protein exit channel in 50S subunit:
Growing protein passes through channel.
exit
exit
50S cut in half and
opened like a book
Protein doesn’t
fold until it’s out
of exit channel.
[E,P,A] three tRNA binding sites on 30S subunit
NH2
[Large subunit]
3 tRNAs
[small subunit]
Three tRNA binding sites on 30S subunit
CCA ends
Exit
Amino-acyl
Peptidyl
Codon-Anticodon
base pairing
Protein Synthesis Cycle: tRNA movements
2
E P A
E P A
1
3
Initiation
complex
formation
E P A
E P A
4
Protein Synthesis Animation:
3-D model (Nierhaus, 2006)
Protein Synthesis Animation
rRNA-mRNA(codon)-tRNA(anticodon) base-pairing:
• another “accuracy” factor
Purine-rich
Start
mRNAs
Initiation: Prokaryotic mRNA-16S rRNA base-pairing
Purine-rich
Start
Prokaryotic initiator fMet-tRNAf
Prokaryotic
initiation complex
[IF2 for AUG start only]
Purine-rich
sequence
[base-pairing]
Elongation
Protein Translation Factors
*
23S rRNA
ribozyme
EF-Tu
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 Factor EF-Tu: Delivers aa-tRNA to ribosome
* (GTP)
Proofreading events can occur on the ribosome
before and after EF-Tu catalyzed GTPase
Correct
1
2
(On the ribosome A-site)
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
Translation Process: Elongation
[Fixed locations of E-P-A]
EF-Tu
5’
3’
23S rRNA
ribozyme
E P A
E P A
GTP
*
GDP + Pi
EF-G
*
[E P
A]
represent
location of tRNAs
E P A
E P A
Translation Process: Elongation
[Fixed locations of E-P-A]
EF-Tu
5’
3’
23S rRNA
ribozyme
E P A
E P A
GTP
*
GDP + Pi
EF-G
*
[E P
A]
represent
location of tRNAs
E P A
E P A
Translocation : EF-G
is an EF-Tu-tRNA mimic
is a G-protein (as is EF-Tu)
E P A
E P A
E P A
E P
New “A”
Next tRNA to A-site
EF-G-GDP off
New “A”
EF-G structure mimics the EF-Tu tRNA complex
Termination:
• Protein release factor(s) recognizes a stop codon.
• Stimulates release of new protein
Termination: tRNA Release Factors
Eukaryotic
[no prokaryotic
structures yet]
is a tRNA mimic
Gly-Gly-Gln “anticodon” may enter
peptidyl transferase center and be involved
in cleavage of peptidyl-tRNA bond.
Ribosome Release Factor (RRF)
• Also a tRNA mimic
• Works with EF-G (and GTP hydrolysis)
• Facilitates separation of tRNA, mRNA, and ribosome subunits.
RRF
CHAPTER 30: PROTEIN SYNTHESIS (Translation)
Translation: mRNA
protein
LECTURE TOPICS
•
Ribosome Structure
•
TRANSLATION PROCESS [Short review]
- Prokaryotic
- Eukaryotic
- Inhibitors [specific steps in process]
Eukaryotic cytoplasmic ribosome (80S)
Eukaryotic Initiation
complex
EIF-2(GTP) for start
AUG only
1) Ribosome binds to cap
Ignore “not fMet”
comments
2) Moves to 1st AUG
EIF2-GDP + Pi
3) Large + small subunits
associate
Elongation
Eukaryotic mRNA: protein-RNA and proteinprotein interactions circularize mRNA
eIF4E binds
to cap
eIF4E binds
to eIF4G
eIF4G binds
to PABI
Antibiotic inhibitors of protein synthesis
*
*
* Common drugs for bacterial infections
Streptomycin: Inhibits
initiation and causes
misreading in
prokaryotic ribosomes
Cyclohehimide (a
common fungicide):
Inhibits eukaryotic
peptidyl transferase
Streptomycin Binds to 30S ribosomal subunit
•
•
Inhibits initiation
Causes misreading of 5` base of codon
Ex: 5`- UUU 3`
Phe
reads as:
5`- AUU 3`
Ile
Puromycin: Terminates protein synthesis
An a.a-tRNA analog that binds to A-site.
Forms peptide bond with
growing protein chain and is
released from ribosome A-site
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
• A chain is a Nglycosidase that
removes A4324 in 28S
rRNA and prevents
elongation factor
binding
Summary
1
2
3
4
5