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Lec 11 Prokaryotic regulation & Yeast Gal

Lec 11 Prokaryotic regulation & Yeast Gal -...

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1 Regulation of Gene Expression DNA mRNA stability 5’ transcriptional initiation, termination translation RNA polymerase post-translational modifications protein degradation
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2 Lactose is broken down into two sugars
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3 lac operon lacZ lacY lacA β -galactosidase P promoter RNA Pol β -galactoside permease β -galactoside transacetylase mRNA Genes involved in lactose utilization are coexpressed from a single promoter. A U G A U G A U G s t o p s t o p s t o p
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4 lacI lacZ lacY lacA lac repressor P O operator RNA Pol Binding of lac repressor to the operator prevents RNA polymerase from binding to the lac operon promoter. In the absence of lactose, lac operon transcription is blocked by the lac repressor .
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5 lacI lacZ lacY lacA P O How does lactose induce expression of the lac operon? 2. β -galactosidase converts lactose into allolactose. low β -gal allolactose basal transcription lactose 3. Allolactose binds lac repressor, causing a conformational change that disrupts binding to operator. 1. Even in presence of repressor, there is a low level of β -galactosidase. 4. Disruption of repressor allows transcription. RNA Pol
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6 Mutations in lac I results in constitutive lac operon expression (expression without inducer). lacI lacZ lacY lacA P O c Mutations in the operator ( O c ) result in constitutive lac operon expression.
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7 Performing complementation tests in bacteria lac + pro + lac - pro - Strains containing F’ elements are partially diploid (merodiploid). A merodiploid can be used to determine whether a mutation is recessive: If this strain is phenotypically Lac + , then the lac mutation is recessive. If this strain is phenotypically Lac - , then the lac mutation is dominant. How do we know that the repressor is a protein and the operator its DNA target?
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8 lac I + is trans-dominant in a merodiploid P lacZ + lacY + lacA + lacI - O main chromosome From this it was deduced that the lac repressor is a diffusible regulator, presumably a protein. lacI + lacZ - lacY - lacA - O P F’ mini- chromosome lacI + lacZ + lacY + lacA + O P lacI - lacZ + lacY + lacA + O P lacI - alleviates repression of all three lac genes in the absence of inducer.
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