Translation - Prokaryotesftp.tugraz.at/pub/Molekulare_Biotechnologie/CHE_167... · 2017-01-17 ·...
Transcript of Translation - Prokaryotesftp.tugraz.at/pub/Molekulare_Biotechnologie/CHE_167... · 2017-01-17 ·...
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Translational coupling
AUG AUG
UAAUAGUGA
AUG
Shine-Dalgarno (SD) SequencerRNA 3‘-GAUACCAUCCUCCUUA-5‘mRNA ....GGAGG..(5-7bp)...AUG
Influences:
Secondary structure!! SD and AUG in unstructured region
Surrounding of SD and AUG!!!
Start
AUG 91%GUG 8UUG 1
Translation - Prokaryotes
Ribosomal protein S1: present only in Gram-negatives (not in Gram-positives): binds to AU-rich sequences found in many prokaryotic mRNAs 15-30 nucleotidesupstream of start-codon
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2 Translation - Eukaryotes
Start Codon
mRNA 5‘-CAP......AUG
Influences:
Surrounding of AUG!!!
Kozak Consensus
.........CCA/GCCAUGG...... mammalian
....... A/TAA/CAA/CAAUGTCT/C........ Yeast
……. gccgcc(A/G)ccAUGG ……….. Wikipedia
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Translation elongation
- Codon usage
- Secondary structures
- Codon structure – translational frameshifting
AAAAAAAAAUCALys Lys Lys Ser
AAAAAAAAAUCALys
Lys Lys Ile
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Taken from: J.E. Krebs, E.S. Goldstein, S.T. Kilpatrick; „Lewin´s Genes XI“; Jones&Bartlett Learning
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Taken from: J.E. Krebs, E.S. Goldstein, S.T. Kilpatrick; „Lewin´s Genes XI“; Jones&Bartlett Learning
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Other mitochondrial codes Other codes in cellular chromosomesUniversal
Codon code Mycoplasma ParameciumEuplotes Yeast Protozoa Mammals
UGA Stop Tryptophan Stop Cysteine Tryptophan Tryptophan TryptophanUAA/UAG Stop Stop Glutamine Stop Stop Stop StopAUA Isoleucine Isoleucine Isoleucine Isoleucine Methionine Methionine MethionineCUA Leucine Leucine Leucine Leucine Threonine Leucine LeucineAGA/AGG Arginine Arginine Arginine Arginine Arginine Arginine Stop
The universal genetic code is used in the chromosomes of most cells, chloroplasts, plant mitochondria, and their viruses and plasmids. A few organisms use slightly different codes in their chromosomes (in the nucleus). The examples of these other nuclear codes are from Mycoplasma (Bacteria) and two different ciii ated protozoa (Eukarya). All nonplant mitochondria use variations of the universal code, whereas plant mitochondria use the universal code. The examples here are only a few of the different types known.1
Universal Triplet Code rare exemptions
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Taken from: J.E. Krebs, E.S. Goldstein, S.T. Kilpatrick; „Lewin´s Genes XI“; Jones&Bartlett Learning
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10 Regulation of Gene Expression
Prokaryotes
Escherichia coli
Lactose Metabolism
Absence of lactose Only few molecules of ß-galactosidase per cell
Presence of lactose about 5000 molecules of ß-galactosidase per cell
Not enzyme is inhibited, enzyme synthesis is affected
Detailed biochemical and genetic analysis
Jacob, Monod, Pardee Nobel prize
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Taken from: J.E. Krebs, E.S. Goldstein, S.T. Kilpatrick; „Lewin´s Genes XI“; Jones&Bartlett Learning
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12 lac-Operon
Ort OOrt I
Taken from: J.E. Krebs, E.S. Goldstein, S.T. Kilpatrick; „Lewin´s Genes XI“; Jones&Bartlett Learning
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Heterogenote analysis
o+ z+
o- z-
o+ z-
o- z+
Cis-configuration
Trans-configuration
inducible
constitutive
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i+ z+
i- z-
i+ z-
i- z+
Cis-configuration
Trans-configuration
inducible
inducible
Heterogenote analysis
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15Model for behaviour of heterogenotes
lacO located adjacent to lacZ, mutation in lacO results in loss
of regulatory function when connected to lacZ,
no complementation by wt-allele in trans
lacI located upstream of lacZ, mutation in lacI results in maintenance
of regulatory function in both configurations to lacZ
complementation by wt-allele
lacO DNA locus, mobile factor binds there and represses synthesis
lacI encodes a mobile factor (= protein) which binds at lacO
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Taken from: J.E. Krebs, E.S. Goldstein, S.T. Kilpatrick; „Lewin´s Genes XI“; Jones&Bartlett Learning
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Φ 80
λ Gene isolationlac operon
Binding studies
Isolation of Lac RepressorlacIq mutant
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Taken from: J.E. Krebs, E.S. Goldstein, S.T. Kilpatrick; „Lewin´s Genes XI“; Jones&Bartlett Learning
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ß-Galactosidase Permease Transacetylase
Inducer: ß-1,6- allolactose(by product of ß-galactosidaseproduced by transglucosylation)
Taken from: J.E. Krebs, E.S. Goldstein, S.T. Kilpatrick; „Lewin´s Genes XI“; Jones&Bartlett Learning
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20 Mutant Oc
Mutation in lacO prevents binding of LacI Repressor protein to Operator
Taken from: J.E. Krebs, E.S. Goldstein, S.T. Kilpatrick; „Lewin´s Genes XI“; Jones&Bartlett Learning
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21 Mutant I -
Mutation in lacI no binding capacity of LacI repressor protein
Taken from: J.E. Krebs, E.S. Goldstein, S.T. Kilpatrick; „Lewin´s Genes XI“; Jones&Bartlett Learning
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Taken from: J.E. Krebs, E.S. Goldstein, S.T. Kilpatrick; „Lewin´s Genes XI“; Jones&Bartlett Learning
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Taken from: J.E. Krebs, E.S. Goldstein, S.T. Kilpatrick; „Lewin´s Genes XI“; Jones&Bartlett Learning
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Taken from: B. Lewin, Essential Genes, Pearson Ed. International
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Taken from: J.E. Krebs, E.S. Goldstein, S.T. Kilpatrick; „Lewin´s Genes XI“; Jones&Bartlett Learning
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Taken from: J.E. Krebs, E.S. Goldstein, S.T. Kilpatrick; „Lewin´s Genes XI“; Jones&Bartlett Learning
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LacI repressor has general low affinity to DNA Unspecific weak binding
LacI repressor has highaffinity to specific operonRegion on DNA Specific strong binding
Taken from: J.E. Krebs, E.S. Goldstein, S.T. Kilpatrick; „Lewin´s Genes XI“; Jones&Bartlett Learning
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Taken from: J.E. Krebs, E.S. Goldstein, S.T. Kilpatrick; „Lewin´s Genes XI“; Jones&Bartlett Learning
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Taken from: J.E. Krebs, E.S. Goldstein, S.T. Kilpatrick; „Lewin´s Genes XI“; Jones&Bartlett Learning
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31 Negative Regulation
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Taken from: J.E. Krebs, E.S. Goldstein, S.T. Kilpatrick; „Lewin´s Genes XI“; Jones&Bartlett Learning
Negative Regulation
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Taken from: J.E. Krebs, E.S. Goldstein, S.T. Kilpatrick; „Lewin´s Genes XI“; Jones&Bartlett Learning
Positive Regulation
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Taken from: J.E. Krebs, E.S. Goldstein, S.T. Kilpatrick; „Lewin´sGenes XI“; Jones&BartlettLearning
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Glucose controls import of lactose
and of other alternative carbonsources
Influence of Glucose on expressionof lac Operon
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Cyclic AMP acts as an inducer
Carbon Catabolite Regulation
CAP (CRP) protein is a positive acting regulator protein
Taken from: J.E. Krebs, E.S. Goldstein, S.T. Kilpatrick; „Lewin´sGenes XI“; Jones&Bartlett Learning
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Taken from: J.E. Krebs, E.S. Goldstein, S.T. Kilpatrick; „Lewin´sGenes XI“; Jones&Bartlett Learning
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Taken from: B. Lewin, Essential Genes, Pearson Ed. International
Regulation at
translation level
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Taken from: J.E. Krebs, E.S. Goldstein, S.T. Kilpatrick; „Lewin´s Genes XI“; Jones&Bartlett Learning
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40 Attenuation
Taken from: J.E. Krebs, E.S. Goldstein, S.T. Kilpatrick; „Lewin´s Genes XI“; Jones&Bartlett Learning
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Taken from: J.E. Krebs, E.S. Goldstein, S.T. Kilpatrick; „Lewin´s Genes XI“; Jones&Bartlett Learning
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Taken from: J.E. Krebs, E.S. Goldstein, S.T. Kilpatrick; „Lewin´s Genes XI“; Jones&Bartlett Learning
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43 Antisense RNA
Taken from: B. Lewin, Essential Genes, Pearson Ed. International
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Taken from: B. Lewin, Essential Genes, Pearson Ed. International
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Taken from: B. Lewin, Essential Genes, Pearson Ed. International
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Taken from: B. Lewin, Essential Genes, Pearson Ed. International
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Taken from: B. Lewin, Essential Genes, Pearson Ed. International
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Taken from: B. Lewin, Essential Genes, Pearson Ed. International
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Taken from: B. Lewin, Essential Genes, Pearson Ed. International
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