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20 Cards in this Set
- Front
- Back
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Transcriptional Regulation Prokaryotes: promoter
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Upstream of RNA coding sequence
Site where RNA pol interacts w/DNA to begin transcription Ensures initiation for that gene always occurs at the same site |
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RNA coding sequence
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DNA strand that is transcribed by RNA Pol into a RNA transcript: template strand-serves as the template for RNA synthesis
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Transcriptional Regulation Prokaryotes: Terminator
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downstream of the RNA coding sequence
Specifies where transcription will stop |
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E. coli Promoter Sequences
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Sequences important for specifying transcription initiation: 35 box (5'-TTGACA-3') and 10 box (5'-TATAAT-3')
RNA Pol Holoenzyme Sigma Factors |
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RNA Pol Holoenzyme
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4 polypeptides (two a, two B, one B') in association with a sigma factor recognize the -35 and -10 regions
Holoenzyme binds nonspecifically to DNA in absence of the sigma factor |
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Sigma factors
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several different types of sigma factors in E. coli
All bind to the core RNA Pol and allow the holoenzyme to recognize different promoters sigma 70: most common, responsible for recognizing most promoters sigma 32: recognizes promoters of heat shock genes sigma 54: fxns in nitrogen starvation conditions sigma 23: fxns in bacteriophage infection |
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How is gene expression regulated?
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Operons: Clustering of genes that are needed at the same time: i.e. the genes are adjacent to each other on the bacterial chromosome
Often transcribed together in a single mRNA molecule: polycistronic mRNA Expression controlled by interaction b/w a regulatory protein and a regulatory site (operator) The operator site is found adjacent to the operon |
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Lactose metabolism: the Lac Operon: discovery
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Francois Jacob and Jacques Monod as well as Josh Lederberg and Andre L'woff form the mid 1940s to the 1960s
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Breakdown of Lactose
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Into glucose and galactose, with B-galactosidase and water
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structural genes of lac operon
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lacZ, lacY, lacA --> transcription
polycistronic mRNA --> translation proteins: b-galactosidase, permease |
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IPTG:
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chemical analog of lactose
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tight regulation of the lac operon
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very low level of enzymes present in cells in the absence of lactose: mRNA has short half life, expression of the operon is turned off when lactose is used up, presence of glucose represses lac operon even when lactose is present in the media
Expression induced by about 1000-fold in the presence of lactose (and absence of glucose): the level of expression is directly proportional to the amount of lactose present in the media |
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The Operon Model- Negative Control of Expression
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Operon Model- cluster of genes whose expression is regulated and expressed as a unit
Absence of lactose: LacI binds to the lacO operator sequence, blocks access of RNA Pol to the operon Presence of lactose: some lactose converted to allolactose (an isomer of lactose): binds to LacI repressor (loses affinity for the operator and falls off) In the absence of the repressor, RNA Pol can bind to the promoter and transcribe the operon |
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allosteric interaction
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allolactose reversibly interacts with the LacI repressor, causing a change in conformation and a change in protein activity
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Mutations that alter experience of the lac Operon
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Mutations in the Structural Genes: lacZ, lacY, lacA, cells with mutations in any of these genes are unable to breakdown lactose
Mutation in Repressor Gene: lacI- constitutive expression of lac operon, lacIs- superrepressor, no expression of lac operon Mutation in Operator Region: lacO- constitutive expression of lac operon |
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genetic regulation of the lac operon
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I- and Oc mutaitons interfere with repressor-operator interactions: allow expression of the lac operon in absence of inducer
Used partial diploid (F' structure) to determine how the I gene and O region function to regulate lac operon expression |
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Effect of lacOc mutation in a merozygote
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phenotype: lacZ is constitutive, but lacY is inducible
Implies that lacOc mutation is cis-acting element and that the lacO region does not encode a diffusible product |
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lacI regulatory mutations
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expression of both operons is repressed in the absence of lactose
expression of both operons is inducible in the presence of lactose: i.e. both lacZ and lacY are expressed This suggests that the lacI gene is trans-dominant to the lacI gene: lacI produces a diffusible cellular product |
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lacIs regulatory mutations
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superrepressor- binds the lacO operator even in the presence of lactose
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Positive Control of the lac Operon- Catabolite Regression Repression
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Glucose is preferred carbon source
If cells are grown in presence of both glucose and lactose, expression is repressed by mechanism of catabolite repression: mediated by catabolite activating protein (CAP) In presence of glucose CAP activity is inhibited resulting in inefficient binding of RNA Pol to the promoter |
