Post on 04-Jun-2018
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Gene Expression: Translation
Chapter 11
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DNA directs the synthesis of proteins
• concept that genes control enzymes
• structure of proteins
• the mechanism of translation
- prokaryotes, eukaryotes
• post-translational modifications to polypeptides
• the genetic code
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Genes control enzymes : Garrod
1. Archibald Garrod (1909):
- individuals with particular homozygous alleles expressed
particular disease, including alkaptonuria and albinism
a defect in a single gene can result
in a metabolic deficiency that causes
an identifiable phenotypic condition
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Genes control enzymes : Beadle and Tatum
Concluded that one gene encoded one enzyme (1941)
current hypothesis: each gene encodes 1 enzyme (or protein)
that functions in a cell to determine a phenotype
Isolated mutant strains of Neurospora that could only grow if
niacin was supplied in culture medium (auxotrophs)- hypothesis : each strain is deficient in 1 enzyme in niacin
pathway
Different intermediates were fed to mutant strains of Neurospora
this positioned the mutants in the niacin biosynthetic pathway
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One enzyme can impact more than
one phenotype
Pleiotropy• Defect in a single gene affects one step in a
biochemical pathway but has multiple effects
- example :mutation that causes tyrosinosis
prevents the degradation of phenylalanine
and tyrosine however, may result in multiple symptoms
(phenotypes): ulcers in corneas, lesion on
skin, mental retardation
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Proteins Are Composed of Amino
Acids
• Amino (NH2) group
• Carboxyl (COOH) group
• Side chains (R groups), four classes:
1. Acidic
2. Basic
3. Nonpolar (hydrophobic)
4. Polar (uncharged)
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The 20 common amino acids
* all side chains are shown at physiological pH (6.8)
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Joining amino acids
Amino acids are joined together by peptide bonds
“dehydration synthesis”
dipolar ion
(zwitterion)
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Protein Structure• Primary: Linear sequence of amino acids
• Secondary: Common folding patterns that form dueto maximization of hydrogen bonds (alpha helix,beta pleated sheet) in peptide backbone of nearby
amino acids (others : extended strands, turns,random coils)
• Tertiary: Overall three-dimensional structure of
protein
• Quaternary: Interaction of more than onepolypeptide to form active protein
** each level of structure depends on the level below it
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Secondary Structure
-helix
-pleated sheet
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Tertiary Structure
Three-dimensional structure ofhuman -globin
forms active site
of an enzyme
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Quaternary Structure
human hemoglobin
-globin
-globin
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Translation Occurs on the Ribosome• Codons (3 base sequences) in mRNA are read sequentially
• tRNAs read the codons using their anticodons• tRNAs have amino acids attached to their 3’ ends
• Amino acids are joined by peptide bonds
• sequence of amino acids in protein is specified by sequence of
codons in mRNA
E. co li
ribosome
A : aminoacyl site
P : peptidyl siteE : exit site
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Aminoacyl-tRNA synthetases
• Covalently attaches (charges) the correct amino acid to 3’ end of the
correct tRNA : recognizes acceptor s tem and ant icodon of tRNA
• Charging is a two-stage reaction in active site of tRNA synthetase:
1. amino acid reacts with ATP (binds AMP, releases PPi)
2. amino acid is detached from AMP and joined to 3’ end of the tRNA
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Decoding Machinery
• 20 different aminoacyl tRNA synthetases
• More than 20 tRNAs
• 64 codons (61 code for a.a. , 3 for STOP codons)
• anticodon of tRNA, and not the amino acid itself,
determines which amino acid is incorporated
result : Ala incorporated into protein
anticodon on tRNA, not amino
acid, is recognized and
dictates which amino acid
is incorporated
anticodon anticodon
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Translation
• Initiation
- formation of ribosome
- recognition of 1st codon
- positioning 1st charged tRNA at P site
• Elongation
- add a.a. from charged tRNAs at A site
• Termination
- recognize STOP codon- stop elongation
- release of polypeptide
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Translational Initiation Must Start
at Correct Codon
• Frameshift mutations: deletion or insertion of one or morebases in mRNA, resulting in shift of reading frame
- If ribosome shifts out of frame it translates different codons,
resulting in a nonfunctional protein
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Initiator methionine in E. coli
(initiator methionine)(internal methionine)
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Initiation of Translation in E. coli
(N-formyl-methionine)
binds at
P site of 30S
IF2 only binds fMet
fMet is the only initiator aa
E : exit site
P : peptidyl tRNA site
A : aminoacyl tRNA site
IF1 binds at A site, prevents 1st
tRNA from binding there
I iti ti f T l ti i E li
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Initiation of Translation in E.coli :
Shine-Dalgarno sequence in mRNA
base pairs with16S rRNA
16S rRNA
mRNA (just upstream of initiator AUG)
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• eIF3 binds 40S subunit
• eIF2-GTP binds Met-tRNA
and then binds 40S subunit
• eIF4 binds 5’ Cap of mRNA
• 5’ Cap of mRNA binds
complex of 40S subunit,
eIF2-GTP, eIF3, and
Met-tRNA
Initiation of Translation in Eukaryotes
Initiator complex forms at 5’ cap :
i
Met
i
Met
GTP
GTP
GTP
PABP i t ti ith i iti t l
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PABP interactions with initiator complexes
enhance translation by helping initiation and
stabilizing mRNApolyA binding protein
http://www.nature.com/horizon/rna/highlights/figures/s2_spec1_f1.html
may also insure that only mature mRNAs are translated
f
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Initiation of Translation in Eukaryotes:
Finding the Start Codon (AUG)
• binding of eIF1 and eIF1Astimulates complex to
scan for AUG
• eIF2-GTP hydrolyzes GTP,
then eIF2-GDP and eIF3
leave complex
• eIF5 allows large
subunit (60S) to join complex
GTP
GTP
GTP
-GDP
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Role of eIF2 proteins
• eIF2 required to bring met-tRNA to small
ribosomal subunit
• eIF2B required to reconstitute the active form
of eIF2 (eIF2-GTP) from eIF2-GDP
i
Met
Mechanisms to Identify Start
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Mechanisms to Identify Start
Codon AUG
Scanning Model
ribosome moves along mRNA until first AUG is reached
• AUG is usually located in Kozak sequence (PuNNAUGG) different from ribosome binding site (5’ Cap)
(ribosome can only bind to 5’ cap)
Mechanisms to Identify Start
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Shunting Model- first AUG is masked in secondary RNA structure or
- first AUG is not in Kozak sequence
ribosome continues to next AUG
Mechanisms to Identify Start
Codon AUG
Mechanisms to Identify Start
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Multiple translation start sitesinternal ribosome entry site (IRES) is used for ribosomeassembly : consensus site; several hundred nucleotides
multiple open reading frames can be translated from 1 mRNA
Mechanisms to Identify Start
Codon AUG
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80S
60S
40S
Met
P A
• A (aminoacyl) site: where incoming charged
tRNA binds ribosome (and reads codon inmRNA)
• P (peptidyl) site: where tRNA with growing
polypeptide chain is positioned
Sites on Eukaryotic Ribosome
Modified from Hyde, Introduction to Genetic Principles
P : peptidyl tRNA site
A : aminoacyl tRNA site
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Translation : Elongation
Binding the charged tRNA at the A site
• Elongation Factors EF-Tu and EF-Ts• GTP
• Charged tRNAs
• Codon of mRNA positioned in A site ofribosome
E. coli :
Binding a Charged tRNA at the A Site
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Binding a Charged tRNA at the A Site
(E. coli )
Charged tRNA
2. EF-Tu-GTP positions
charged tRNA by binding A siteof ribosome if codon binding is correct
1. Charged tRNA binds EF-Tu-GTP
3.
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Translation: ElongationPeptide Bond Formation
• Peptidyl transferase is in the ribosomal RNA of largesubunit of the ribosome (here RNA is the enzyme ; peptidyltransferase is a r ibozyme )
- catalyzes :
1) cleavage of high-energy bond between amino acid
and tRNA in the P site
2) formation of a peptide bond between the a.a. attached
to tRNA in the A site and the a.a attached to tRNA in
the P site
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Translation: Elongation
T l ti El ti
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Translation: Elongation
Ribosome Translocation :
1. Polypeptide on tRNA inP site is transferred toa.a. on charged tRNA in
A site
2. EF-G enters at A site ;hydrolyzes GTP
3. Ribosome moves down
mRNA one codon4. Uncharged tRNA in P
site moves to E site and
exits ribosome
Cycle of peptide bond formation
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Cycle of peptide bond formation
and translocation on ribosome
Cycle of peptide bond formation
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Cycle of peptide bond formation
and translocation on ribosome
(1)
Translation : Elongation
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Translation : Elongation
(Eukaryotes)
• eEF1 instead of EF-Tu
• eEF1 instead of EF-Ts• eEF2 instead of EF-G
• no E site on ribosome
very similar to E.coli, except :
80S
60S
40S
Met
P A
Binding a Charged tRNA at the A Site
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Binding a Charged tRNA at the A Site
(Eukaryotes)
eEF1 -GTP positions
charged tRNA by binding A siteof ribosome
eEF1 eEF1
eEF1
eEF1
eEF1
eEF1
eEF1
no E site in ribosome
1. Charged tRNA binds eEF1 -GTP
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Translation : Elongation
Error rate : 1 in 10,000 amino acids incorporated into protein
Speed of a.a. incorporation : 15 a.a. per second (E. coli ) 300 a.a. protein made in 20 s !
2-5 a.a. per second (eukaryotes)
300 a.a. protein in 1-2.5 min !
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Translation: Termination (E. coli )
Insert Fig.
11.29molecular mimicry : a protein
resembles the shape of ananticodon in a tRNA
Translation: Termination
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Translation: Termination
(eukaryotes)
very similar to E.coli except :
• eRF1 instead of RF1 or RF2
• eRF3 similar to RF3
involved with peptide release, GTP hydrolysis
needed
E coli translation is inhibited by
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E. coli translation is inhibited by
antibiotics
• azithromycin, erythromycin
block peptide exit tunnel ; prevent elongation
• streptomycin bind to A or P sites and induce errors in
bacterial translation
• tetracycline
block binding of charged tRNA to A site
E coli translation is inhibited by
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E. coli translation is inhibited by
antibiotics
30S subunit 50S subunit
Poehlsgaard and Douthwaite, Nature Reviews : Microbiology 3:870-881 (2005)
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Energy use in translation
4 high energy phosphate bonds are used per peptide
bond
• 2 high energy phosphate bonds to charge tRNA(tRNA synthetase, ATP AMP)
• 1 high energy phosphate bond to bind the A site(EF-Tu or eEF1 , GTP GDP)
• 1 high energy phosphate bond to translocate (EF-Gor eEF2, GTP GDP)
~90% of bacterial energy production goes into
protein synthesis !
Comparisons : bacterial and
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Comparisons : bacterial and
eukaryotic translation
• Prokaryotic mRNA can contain more than one gene per
transcript : polycistronic
• Eukaryotic mRNA contains one gene, monocistronic
- ribosome binds initiation factors recognizing the 5’ cap
Transcription and Translation Are
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Transcription and Translation Are
Coupled in E. coli
• eukaryotes : transcription and translation are uncoupled
- transcription : nucleus
- translation : cytoplasm
Polysomes: Many Ribosomes
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Polysomes: Many Ribosomes
Bound to mRNA
3’
5’
occur in prokaryotes and eukaryotes
Functional Sites on a Ribosome
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Functional Sites on a Ribosome
Growing
polypeptide
mRNA
EF-Tu site
EF-G site
Peptidyl
transferase site
What happens to a
polypeptide once it is
translated on a ribosome ?
- depends on location of
ribosome :
1) free in cytoplasm
2) associated with ER
Targeting mRNA to the
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Targeting mRNA to the
Endoplasmic Reticulum
1. Signal peptide: a positively charged a.a.followed by 10-15 hydrophobic amino acids
2. Bound by Signal Recognition Particle (SRP),
halts translation (SRP : 6 proteins and a 7S RNA)
3. SRP binds docking protein on endoplasmic
reticulum
4. Protein translation resumes through
translocation channel (translocon) in ER
5. Signal peptide is removed by signal
peptidase in endoplasmic reticulum
Targeting proteins to ER
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Targeting proteins to ER
protein is either secreted from cell or inserted into the membrane
T ti b t i t ER
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Targeting membrane proteins to ER
http://kc.njnu.edu.cn/swxbx/shuangyu/4.htm
Transporting membrane proteins to cell surface
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Transporting membrane proteins to cell surface
http://kc.njnu.edu.cn/swxbx/shuangyu/4.htm
P tt l ti l Ch t P t i
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Posttranslational Changes to Proteins
1. Protein folding
- some proteins spontaneously fold into correct tertiary
structure
- others require chaperones that facilitate proper
folding2. Cleavage of amino terminus : secreted or membrane
proteins
3. Phosphorylation of polypeptide : consensus sequence
4. Addition of sugars or carbohydrates to some R groups
5. Lipid addition
- allows membrane attachment without a signal sequence
* posttranslational modifications : more common in eukaryotes
Ch
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Chaperones
- unfold partially folded
protein, allow it to fold in a
different way
- specific to different classes
of proteins
- GroE
GroEL (Hsp60)
GroES (Hsp10)
- Hsp90
- Hsp70
Posttranslational Changes to Proteins
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http://www.sciencedirect.com/science/article/pii/S0959437X09000756
http://www.piercenet.com/
http://www.ideacenter.org/contentmgr/showdetails.php/id/838
Phosphorylation
Lipid Addition
Carbohydrate Addition
Posttranslational Changes to Proteins
G ti C d
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Genetic Code
• Codons are 3 nucleotide units
- smallest number that can code for 20 a.a.
• Nonoverlapping
• No punctutation (no separation between codons)
• Redundant/degenerate
- more than one codon for some amino acids
• Elucidated using artificially synthesized RNAs (i.e.,
poly U) or trinucleotides – Added to tube containing cell free system
– Asked: What amino acids were joined together in this cell
free system using these RNA templates?
Codons Are Read Three Bases at a Time
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Codons Are Read Three Bases at a Time
• Insertion of one
base shifts reading
frame
• Insertion of
two more bases
restores correctreading frame
- insertion or deletion of multiples of 3 nucleotides restored
wild-type function of mutated protein
3 nucleot ides code for 1 am ino acid
Francis Crick :
- mutated viral protein with proflavin : adds or deletes 1 nucleotide
Genetic Code Is Nonoverlapping
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Genetic Code Is Nonoverlapping
• position of a.a. next
to each other in
protein indicated
nonover lapping
structure
• no pun ctuat ion
(noncoding nucleotides
between codons)
The Genetic Code
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The Genetic Code
Crick, Nirenberg, Matthaei, Leder :
1. mutant phage
2. synthetic mRNAs in cell-free system
- polynucleotide repeats
- dinucleotide repeats
3. synthetic codons
- filter binding assay
tRNA Anticodon Base Pairing with
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gmRNA Codon
mRNA and tRNA strands are antiparallel
tRNA
mRNA
“Wobble” in tRNA/mRNA Base
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Pairing
• 64 possible codons (3 are stop codons)
• only 50 tRNAs in E. coli
* 3rd base of codon in mRNA and 1st base ofanticodon in tRNA can “wobble”
– base pairing does not have to be exact at this
position
Allows different codons (which differ only in
third position) to be read by same tRNA
Base Pairing Possibilities at Wobble Position
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Base Pairing Possibilities at Wobble Position
Insert Fig.
11.45
Universality of Genetic Code
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Universality of Genetic Code
• Shared by most living organisms• Some codons are read differently in
– Yeast mitochondrial genes
– Drosophila
– Higher plants
– Mycoplasmas
– Ciliated protozoa
– Site specific variation : interpretation ofcodon depends on its location
E. coli : GUG and UUG sometimes used as
initiator methionine
Design of Genetic Code
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Design of Genetic Code
* genetic code is designed to minimize
impact of mutations
– mutations in 3rd position of codon are not likely
to change encoded amino acid- ACU, ACC, ACA, ACG all code for Thr
– functionally related amino acids are encoded bysimilar codons
- all codons with U as middle base encodehydrophobic amino acids (Phe, Leu, Ile, Val)
genetic code is not random