translation. translation- the synthesis of protein from an rna template. five stages: preinitiation...
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Translation
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Translation
Translation- the synthesis of protein from an RNA template.
Five stages:
Preinitiation
Initiation
Elongation
Termination
Post-translational modification
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Translation Is the Most Complicated Biological Process Known
In eukaryotes,>70 ribosomal proteins20 (more) proteins to activate aa’s12 (more) auxiliary enzymes≥100 proteins for processing≈40 tRNA and rRNAs (minimum)Other specific proteins ~300 molecules involved
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Translation
• Importance
• Location: ribosomes (predominantly Cp)
• Highly regulated
• Fast: ~20 aa/sec
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Functions of the Types of RNA
mRNA- serves as a template code
tRNA- serves as an adapter molecule
rRNA- holds molecules in the correct position, protein portion also catalyze reactions
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The Genetic Code has been Cracked
Poly U codes for poly Phe
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The Genetic Code has been CrackedTriplet Codons Code for a Single Amino
AcidUUU = Phe
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The Genetic Code has been CrackedThe Code is Commaless
AUGUUU
Met Phe
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The Genetic Code has been CrackedThe Code is Nonoverlapping
AUGUUU AUGUUU
Met Phe Not Met Cys Val
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The Genetic Code has been CrackedThe Code is Redundant
1 codon for Met, Trp
all other amino acids have 2-6 triplets
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The Genetic Code has been Cracked The Code is Degenerate
UCU Ser
UCC Ser
UCA Ser
UCG Ser
“Wobble” exists in the third position
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The Wobble Hypothesis
• First two bases of codon form strong h-bonds• If 1st base of anticodon is C or A (i.e. last base
of codon is G or U), only one:CG and AU base pairs formed
• If 1st base of anticodon is G or U two codons can be recognized: U to A or G; G to C or U
• If 1st base of anticodon is I, three codons recognized: I to A, U or C
• By not having to verify all three bases, speed up process
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The Genetic Code has been Cracked The Code is Unambiguous
UUU doesn’t code for Phe sometimes, and other times Ser
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The Genetic Code has been Cracked The Code is Universal
Not completely true
Mitochondria, e.g., have some codons different
Nuance is codon usage
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The Genetic Code has been Cracked Three Reading Frames are Possible
ACUGUCGCUC...
One: ACU GUC GCUC...
Two: CUG UCG CUC...
Three: UGU CGC UC...
(Four):GUC GCU C...
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The Genetic Code has been Cracked Embedded Genes are Possible
Fig. 26-3 Lehninger POB 4th Ed.
Open Reading Frame- ORF a long enough sequence between start and stop to code for legitimate protein.
In viral systems, there are also examples of overlapping genes.
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The Genetic Code has been Cracked Nonsense Codons are Stop Points
UAA, UAG, and UGA
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There is a 5’UTR
Shine-Dalgarno sequence (prokaryotes)
~10 nt upstream of initiation codon
Consensus: 4-9 Pu 8-13 nt upstream
Positions ribosome at correct start site
mRNA Structure
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All tRNA’s Have a Similar but not Identical Structure: “Cloverleaf”
• ~75 nt• Acceptor arm- CCA-3’• TC arm• Variable length extra
arm ~3-~20 nt• anti-codon arm• D arm• canonical positions• Identity elements
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rRNA Structure (E. coli Because Well-Known)
• Small subunit (30S)– 16S RNA– 21 proteins (S1-S21)
• Large subunit (50S)– 5S and 23S RNA– 36 proteins (L1-L36)
• Combined = 70S
• Eukaryote 40S + 60S = 80S
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Components of Mammalian Ribosomes(After Harper’s Biochemistry)
Component Mass Protein RNA
No. Mass Size Mass Bases
40S s/u 1.4x106 ~35 7x105 18S 7x105 1900
60S s/u 2.8x106 ~35 7x105 18S 7x105 1900
~50 1x106 5S 35,000 120
5.8S 45,000 160
28S 1.6x106 4700
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rRNA StructureScientific American circa 1960 and Science 2002
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The P Site and the A SiteFig. 27-11 Lehninger POB 3rd Ed.
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The Preinitiation Stage is Characterized by Formation of Required Starting Complexes
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Preinitiation - Charging the tRNA
OCH2
OH OH
NN
N N
NH2
O
P
O
OCCtRNA
C H
R
H3N
C O
OCH2
O OH
NN
N N
NH2
O
P
O
OCCtRNA
ATP PPi
CO2
C H
R
H3N
AMP
Aminoacyl-tRNAsynthetase
.
.
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Aminoacyl-tRNA Synthetase
• One for each amino acid• 2 step mechanism:
1. attach a.a. to AMP
2a. transesterify to 3’ (class 2)
2b. transesterify to 2’ and then rearrange(class 1)
• Proofread– identity elements– “sieve”
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Mechanism of Class I vs. Class IIFig. 27-14 Lehninger POB 4th Ed.
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Perspective on ScaleFig. 27-17(a) Lehninger POB 4th Ed.
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Preinitiation: Formylation of met-tRNAfmet (Prokaryotes Only)
met-tRNAfmetfmet-tRNAfmetFormyltransferase
formyl-THF THF
.
.
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Preinitiation: Dissociation of Ribosomes (IF-1 and IF-3)
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Preinitiation: IF-2:GTP Binary Complex Formation
• IF-2B represents a guanine exchange molecule
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Preinitiation: IF-2:GTP:Charged tRNA Ternary Complex Formation
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Preinitiation: 40S Preinitiation Complex
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Initiation: IF-4F, 4A and 4B Bind mRNA to Place it on Small Subunit
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Initiation: 40S Initiation Complex
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Initiation: 80S Initiation Complex
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Initiation
Preinitiation complexes form an 80S complex:
small subunit, ternary complex (GDP + Pi leave), mRNA, large subunit, aminoacyl tRNA
P-site- only thing that can enter is a peptide
In prokaryotes, f-met “tricks” the ribosome
A-site- only thing that can enter is an aminoacyl tRNA
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Elongation
1. EF-1:GTP:aminoacyl-tRNA ternary complex enters A-site; GDP + Pi leave
(EF-Tu and EF-Ts involved with GTP metabolism in prokaryotes)
2. Peptide bond forms as P-site content is transferred onto A-site occupant
3. Translocation requires GTP-bound EF-2
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Elongation I
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Elongation: Peptide Bond Forms as P-site Content is Transferred Onto A-Site
Occupant
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Mechanism of Peptide Bond Formation
OO C
O
C
NH2
H
R1
OHA
tRNA CC
OO C
O
C
NH2
H
R2
OHA
tRNA CC
P Site
A Site
.
.
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Mechanism of Peptide Bond Formation
OOH
OHA
tRNA CCP Site
A Site
C C
NH3
H
R1O
OO C
O
C
N
H
R2
OHA
tRNA CCH
.
.
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Elongation: Translocation Requires GTP-bound EF-2
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Termination
1. UAA, UAG, UGA is enveloped by A-site of ribosome
2. RF-1 enters A site
3. GTP is hydrolyzed, H2O is used to cleave protein off tRNA
4. Components are recycled to synthesize another protein molecule
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Termination: UAA, UAG, or UGA is Enveloped by A-site of Ribosome
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Termination: RF-1 Enters A Site
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Termination: GTP is Hydrolyzed and H2O is Used to Cleave Protein Off tRNA
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Energetics
Each amino acid residue requires >4 ATP equivalents
ATP→AMP + PPi to “charge” tRNA
1 GTP is used to place aminoacyl-tRNA into A-site
1 GTP is used to translocate after each peptide bond formation
ATP hydrolysis for unknown purpose at…
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Regulation of Translation: eEF 2
a. eEF 2 phosphorylated under stress
b. when phosphorylated, eEF 2B doesn’t allow GDP-GTP exchange and protein synthesis stops
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Regulation of Translation: eIF 4F
4F is complex of 4E (cap binding protein) and 4A (ATPase that unwinds RNA) and 4G (function unknown)
a. eIF-4E can be phosphorylated: why or how unknown- but this activates
b. 4E-BP complex forms which inactivates 4E. Phosphorylation in presence of insulin dissociates complex
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Post-translational Modifications
1. Proteolytic cleavage (most common)Signal sequences; zymogens
2. Disulfide bond formation: not as well understood3. Group addition
a. Glycosylation (most complex known)b. Acetylation or phosphorylation, etc.c. Farnesyl or Geranylgeranyl
4. Amino acid modificationa. Hydroxylation of Pro (in ER)b. Methylation of Lys
5. Other things
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Signal Sequence:Human PreProInsulin
Met Ala Leu Trp Met Arg(+) Leu Leu Pro
Leu Leu Ala Leu Leu Ala Leu Trp Gly Pro
Asp Pro Ala Ala Ala Phe Val
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SRP Ribosome CycleFig. 27-33 Lehninger POB 4th Ed.
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Other Signal SequencesFig. 27-39 Lehninger POB 3rd Ed.
• KDEL anywhere in protein directs back to ER
• Chloroplast and Mitochondria:– Different in that whole
protein is made and then directed via chaperone proteins
– Different sequences: mitochondrial not contiguous
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Other Signal SequencesFig. 27-37 Lehninger POB 4th Ed.
• KDEL anywhere in protein directs back to ER
• Chloroplast and Mitochondria:– Different in that whole protein
is made and then directed via chaperone proteins
– Different sequences: mitochondrial not contiguous
• Nuclear signal:– Different in that not cleaved
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Other Signal Sequences
• KDEL anywhere in protein directs back to ER
• Chloroplast and Mitochondria:– Different in that whole protein
is made and then directed via chaperone proteins
– Different sequences: mitochondrial not contiguous
• Nuclear signal:– Different in that not cleaved
• Doesn’t have to be protein signal: M6P targets to lysosome
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Other Proteolytic Events
• Met aminopeptidase
• Proprotein vs preproprotein
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Disulfide Bond Formation is Not Well Understood
• Occurs in ER
• PDI and Chaperones involved
• PDI “shuffles” disulfide bonds
• Chaperones consist of two rings of identical s/u’s and somehow facilitate appropriate interactions
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Glycosylation Occurs in Multiple Compartments (ARB figure)
Oligo “tree” transferred en bloc from dolichol-based structure
Some trimming takes place before export from ER
Often, further trimming in cis-Golgi…
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Glycosylation Continued
…before building up with different sugars takes place
Packaging occurs as the proteins pass through the trans-golgi network
Exocytosis into bloodstream takes place
Microheterogeneity
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Lipid MoietiesFig. 27-30 Lehninger POB 4th Ed.
• CaaX farnesylation signal (a for aliphatic)• aaX removed after farnesyl added• CXX or CXC geranylgeranylation signal (X for
anything)
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Protein Degradation: The Ultimate in Posttranslational Modification
Regulated:
1. The N-end Rule
not a sensitive system
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The N-End Rule
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Protein Degradation
Regulated:
1. The N-end Rule
not a sensitive system
2. Lysosome
endocytosis3. The Ubiquitin system
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UbiquinationFig. 27-41 Lehninger POB 4th Ed.
• Ubiquitin common throughout the animal kingdom
• 76 aa (8.5 kDa) i.e. small• 53% homology between yeast
and humans
• E1 (ubiquitin activating enzyme)• E2 (ubiquitin carrier protein)• E3 (ubiquitin protein ligase) selects proteins to tag by N-term
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More Than You Ever Wanted to Know About UbiutinScience 315: 201-205, 2007
Monoubiquitination- a single Ub is attachedMultiubiquitination- several residues of target
protein each have single Ub is attachedPolyubiquitination- numerous copies of Ub attached
to same site of target protein. Ub has 7 different Lys, so many ways to do this. Once polyUb’d, targeted for destruction
Ub can be removed from proteinSo clearly there must be mechanism to determine
level of Ub
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Ub is Now Known to Do More Than Just Target for Destruction
Science 315: 201-205, 2007
• In yeast, monoUb is sufficient as an endocytic internalization signal (less clear in animals)– necessary, sufficient
• When epidermal growth factor receptor (EGFR) was stimulated at low epidermal growth factor (EGF) concentrations in HeLa cells, EGFR ubiquitination was not detected, and the receptor localized with clathrin; however, at high EGF concentrations, EGFR was ubiquitinated, and the receptor localized with both caveolae and clathrin.
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Ub is Now Known to Do More Than Just Target for Destruction
Science 315: 201-205, 2007
• Endocytosis of certain membrane proteins seems to require polyubiquitination. For example, in the case of the ß2AR, (recall this is a G protein–coupled receptor), agonist stimulation leads to rapid polyubiquitination of both the receptor and the receptor regulatory protein ß-arrestin.
• ...anthrax toxin triggers ubiquitination of its receptor to facilitate efficient and rapid endocytosis of the toxin-receptor complex. ... important for toxin action, because passage through low-pH endosomal compartments makes the toxin competent to induce toxicity in cells
• Protein ubiquitination is best compared to protein phosphorylation.
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Ub is Now Known to Do More Than Just Target for Destruction
Science 315: 201-205, 2007