Translation: overview The genetic code
Transcript of Translation: overview The genetic code
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TRANSCRIPTION RNA is transcribedfrom a DNA template.
DNA
RNApolymerase
RNAtranscript
RNA PROCESSING In eukaryotes, theRNA transcript (pre-mRNA) is spliced andmodified to producemRNA, which movesfrom the nucleus to thecytoplasm.
Exon
Poly-A
RNA transcript(pre-mRNA)
Intron
NUCLEUSCap
FORMATION OFINITIATION COMPLEX
After leaving thenucleus, mRNA attachesto the ribosome.
CYTOPLASM
mRNA
Poly-A
Growingpolypeptide
Ribosomalsubunits
Cap
Aminoacyl-tRNAsynthetase
AminoacidtRNA
AMINO ACID ACTIVATION
Each amino acidattaches to its proper tRNAwith the help of a specificenzyme and ATP.
Activatedamino acid
TRANSLATION A succession of tRNAsadd their amino acids tothe polypeptide chainas the mRNA is movedthrough the ribosomeone codon at a time.(When completed, thepolypeptide is releasedfrom the ribosome.)
Anticodon
A CC
A A AUGGUU UA UG
U ACE A
Ribosome
1
Poly-A
5′
5′
3′
Codon
2
3 4
5
From Gene to Phenotype- part 3DNA
mRNA
polypeptide
Lecture Outline 11/9/05• Review translation:
– Initiation, elongation, termination– EPA model
• Post-translational modification of polypeptides• Signal sequences• Mutations (again)
Exam 3 is next Monday. It will cover mitosis andmeiosis, DNA synthesis, transcription, translation,genetics of viruses.(chapters 12, 13, 16, 17, part of 18 (to page 345))
Translation: overviewTRANSCRIPTION
TRANSLATION
DNA
mRNARibosome
Polypeptide
Polypeptide
Aminoacids
tRNA withamino acidattachedRibosome
tRNA
Anticodon
mRNA
Trp
Phe Gly
A G C
A A A
CC
G
U G G U U U G G C
Codons5′ 3′
The ribosome is themachine that builds thepolypeptide
tRNA serves asan “adaptor”that brings thecorrect aminoacid to eachcodon.
The genetic codeSecond mRNA base
U C A G
U
C
A
G
UUU
UUCUUAUUG
CUUCUCCUACUG
AUUAUCAUAAUG
GUUGUCGUAGUG
Met orstart
Phe
Leu
Leu
lle
Val
UCU
UCCUCAUCG
CCUCCCCCACCG
ACUACCACAACG
GCUGCCGCAGCG
Ser
Pro
Thr
Ala
UAUUAC
UGU
UGCTyr Cys
CAUCACCAACAG
CGUCGCCGACGG
AAUAACAAAAAG
AGU
AGCAGAAGG
GAUGACGAAGAG
GGUGGCGGAGGG
UGG
UAAUAG Stop
Stop UGA Stop
Trp
His
Gln
Asn
Lys
Asp
Arg
Ser
Arg
Gly
U
CA
GUCAG
UCAG
UCAG
Firs
t mR
NA
bas
e (5′ e
nd)
Third
mR
NA
bas
e (3′ e
nd)
Glu
U
C
A
G
U C A GUCAGUCAGUCAGUCAG
3′ACCACGCUUAAGACACCU
GC *
GU GUCU
GAGGU
A
A A GUC
AGACC
CGAGAG GG
GACUCAUUUAGGCG5′
Hydrogenbonds
*
*
**
*
**
*
* **
5’-AUGCAAUUCGGAAAC
Codon in the mRNA
2
4
An aminoacyl-tRNA synthetase joins aspecific amino acid to a tRNA
Amino acid
ATP
Adenosine
Pyrophosphate
Adenosine
Adenosine
tRNA
P P P
P
P Pi
PiPi
P
AMP
AppropriatetRNA bonds to amino
Acid, displacingAMP.
Active site binds theamino acid and ATP. 1
3
Aminoacyl-tRNAsynthetase (enzyme)
Activated amino acidis released by the enzyme.
Each tRNA has aslightly differentshape
How does the ribosome findAUG?
• Prokaryotes have a special bindingsequence upstream of the start codon.
• In Eukaryotes,the ribosome binds to the5’ cap and “scans” the message for anAUG.
See the Animation
• www.dnai.org
Inhibition of protein synthesisToxin Mode of action Target
Puromycinforms peptidyl-puromycin, prevents
translocationProcaryotes
Tetracyclineblocks the A-site, prevents binding of aminoacyl
tRNAsProcaryotes
Chloramphenicol blocks peptidyl transfer Procaryotes
Cycloheximide blocks peptidyl transferase Eucaryotes
Streptomycin inhibits initiation at high concentrations Procaryotes
Diphtheria toxin catalyzes ADP-ribosylation of residue in eEF2 Eucaryotes
Erythromycin binds to 50S subunit, inhibits translocation Procaryotes
Ricininactivates 60S subunit, depurinates an
adenosine in 23S rRNAEucaryotes
NOTE: Prokaryotes (this generally includes proteinsynthesis in mitochondria and chloroplasts)
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Only the anticodon of tRNA determineswhich amino acid is added by a
ribosome.
• Experimental evidence:– Convert cystein to alanine chemically, after
it is attached to tRNA (remove SH group)– Alanine shows up in Cystein sites
The amino acid carried by a tRNA isindependent of the anticodon sequence
• Determined by the amino-acyl tRNAsynthetase enzyme– tRNA with mutations in the anticodon still
have their normal amino acid at the 3’end.
– Experiment:. mutate anticodon of tRNAthr
(AGU-->AGG)• Now binds to proline codon instead (CCU).• Those tRNA still carry threonine, but now
bind to proline sites.• Threonine inserted into polypeptide where
proline normally goes.
Glycine doesn’t fit . .
Alananine tRNA synthetaseAminoacyl tRNAsynthetaseenzyme isspecific to aparticular aminoacid and aparticular tRNA
Quality control
• Both cap and tail bind to initiation factors tostart translation– Ensures that mRNA is intact
• Small subunit can detect mis-paired tRNAand remove them– Needs a short delay before peptide bond is formed
(to give time for proofreading)
• Error rate: about 10-4
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Cost of protein biosynthesis
• Synthesis of aminoacyl tRNAs 2 ATPs• Formation of 1 peptide bond 2 GTPs
– 1 for codon recognition; 1 for translocation
• Proofreading 1 ATP/error
• Construction of a specific amino acid sequence ismuch more costly than formation of a randompeptide bond!
Transcription and translation canoccur simultaneously
DNA
Polyribosome
mRNA
Direction oftranscription
0.25 µmRNApolymerase
Ribosome
DNA
mRNA (5′ end)
RNA polymerase
Polypeptide
Post translationalmodifications and sorting
Glycosylation
Signal directs protein to the right compartment
The signal mechanism for targetingproteins to the ER
Foldsto finalshape
Translationbegins inthe cytosol
SRP binds to the signal peptide,
Attaches totranslocationpore in ERmembrane
Polypeptidesynthesizedinto the ER
Signalpeptideremoved
1 2 3 4 5 6
Ribosome
mRNASignalpeptide
Signal-recognitionparticle(SRP) SRP
receptorprotein
Translocationcomplex
CYTOSOL
Signalpeptideremoved
ERmembrane
Protein
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Brooker Figure 13.22
Destined for ERDestined for cytosolor other organelles
Importedduringtranslation
Importedaftertranslation
Signal peptidedetermines where itgoes
Stays within themembranesystem
Chaperones help fold proteinsHsp 70 covers exposedhydrophobic patches untilthe protein can fold
Hsp60 is like an isolation chamber
Mis-folded proteins aremarked for destruction with
ubiquitinUbiquitin tail
Proteosome acts as garbage disposal
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Mutations (again)
The molecular basis of sickle-celldisease: a point mutation
In the DNA, themutant templatestrand has an A where the wild-type template has a T.
The mutant mRNA has a U instead of an A in one codon.
The mutant (sickle-cell) hemoglobin has a valine (Val) instead of a glutamic acid (Glu).
Mutant hemoglobin DNAWild-type hemoglobin DNA
mRNA mRNA
Normal hemoglobin Sickle-cell hemoglobin
Glu Val
C T T C A T
G A A G U A
3′ 5′ 3′ 5′
5′ 3′5′ 3′
Base-pair substitutionWild type
A U G A A G U U U G G C U A AmRNA 5′Protein Met Lys Phe Gly Stop
Carboxyl endAmino end
3′
A U G A A G U U U G G U U A A
Met Lys Phe Gly
Base-pair substitutionNo effect on amino acid sequence
U instead of C
Stop
A U G A A G U U U A G U U A A
Met Lys Phe Ser Stop
A U G U A G U U U G G C U A A
Met Stop
Missense A instead of G
NonsenseU instead of A
Base-pair insertion or deletionmRNAProtein
Wild type
A U G A A G U U U G G C U A A5′
Met Lys Phe Gly
Amino end Carboxyl end
Stop
Base-pair insertion or deletionFrameshift causing immediate nonsense
A U G U A A G U U U G G C U A
A U G A A G U U G G C U A A
A U G U U U G G C U A A
Met Stop
U
Met Lys Leu Ala
Met Phe GlyStop
MissingA A G
Missing
Extra U
Frameshift causing extensive missense
Insertion or deletion of 3 nucleotides:no frameshift but extra or missing amino acid
3′
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Mutations in the 3rd positionof a codon are often silent
Second mRNA baseU C A G
U
C
A
G
UUUUUCUUAUUG
CUUCUCCUACUG
AUUAUCAUAAUG
GUUGUCGUAGUG
Met orstart
Phe
Leu
Leu
lle
Val
UCUUCCUCAUCG
CCUCCCCCACCG
ACUACCACAACG
GCUGCCGCAGCG
Ser
Pro
Thr
Ala
UAU
UACUGUUGC
Tyr Cys
CAUCACCAACAG
CGUCGCCGACGG
AAUAACAAAAAG
AGU
AGCAGAAGG
GAUGACGAAGAG
GGUGGCGGAGGG
UGG
UAAUAG Stop
Stop UGA Stop
Trp
His
Gln
Asn
Lys
Asp
Arg
Ser
Arg
Gly
U
CA
GUCAG
UCAG
UCAG
Firs
t mR
NA
bas
e (5′ e
nd)
Third
mR
NA
bas
e (3′ e
nd)
Glu
For amino acids thathave only two codons,the 3rd base will eitherboth be purines or bothbe pyrimidines
Wobble in 3rd position