Sequence dna mutations identifying regulatory genes

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Unformatted text preview: ls adjacent lac genes lac • OC operator mutation is cisoperator cis dominant Lewin Genes VII 2000 Oxford University Press OC mutation first evidence of a regulatory DNA sequence DNA MUTATIONS IDENTIFYING REGULATORY GENES & SEQUENCES REGULATORY THE MECHANISM OF REPRESSION O3 O2 O1 Lac repressor binds as a tetramer to two operators, O1 and upstream auxiliary one, O3. THE MECHANISM OF LAC REPRESSION Two competing hypotheses explain the mechanism of repression of the lac operon: 1)RNAP can bind to the lac promoter in the presence of repressor, but the repressor inhibits the transition from abortive transcription to processive transcription. 2) The repressor, by binding to the operator, blocks access by the RNAP to the adjacent promoter. In addition to the classical (major) lac operator adjacent to the promoter, two auxiliary lac operators exist: one each upstream and downstream. All three operators are required for optimum repression, two work reasonable well, but the classical/major operator (O1) by itself produces only a modest amount of repression. POSITIVE OPERON REGULATION BY cAMP-CAP • Many operons respond to carbon nutrient conditions Many glucose preferred energy source • cAMP responds to glucose concentration • Glucose drops, cAMP rises • Catabolite activating protein (CAP) with cAMP binds to the Catabolite activator site and helps RNAP to form an open promoter complex complex THE LAC CONTROL REGION The CAP-binding sites in the lac, gal, and ara operons all contain a conserved sequence TGTGA, suggesting it is an important part of the CAP-binding site. The lac operon have remarkably weak promoters. Their -35 boxes are particularly unlike the consensus sequences; in fact, they are almost unrecognizable. Why? If the lac operon had a strong promoter, RNAP would form open promoter complexes readily without help from CAP and cAMP, and it would therefore be active even in the presence of glucose. CATABOLITE REPRESSION CATABOLITE • • • Catabolite repression: system that exercises glucose preference by inhibiting expression of operons encoding enzymes of other metabolic pathways (e.g. lac, gal & ara operons) metabolic lac gal ara Model: CAP-cAMP dimer binds activator site & interacts with CTD Model: of the α RNAP subunit and promotes the formation of the open promoter complex promoter So, for high levels transcription from lac operon we need both So, lac inducer (lactose) and CAP-cAMP inducer CAP-cAMP BENDS DNA BENDS • CAP DN...
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