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G lac repressor bound by lac 6 lactose inducer which

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Unformatted text preview: inducer: a small operon inducer small molecule that causes the production of enzymes needed 1 1 4 to metabolize it to • E.g. lac repressor bound by lac 6 lactose inducer, which leads to 45 lacZYA expression & lactose lacZYA Weaver 2002 Molecular Biology Ed. 2 3 cleavage cleavage • gratuitous inducers: inducer - a side reaction by β -galactosidase side -galactosidase molecules that are not not rearranges lactose to allolactose, metabolites but stimulate stimulate which is the actual inducer that transcription transcription binds the lacI repressor lacI • E.g. IPTG induces lacZYA IPTG lacZYA transcription (used to induce lacZ reporter in vectors such as lacZ Bluescript) Bluescript) FEATURES OF lac OPERON lac INDUCTION INDUCTION • lac operon: low basal transcription, rapid induction rapid • lac mRNA unstable: t1/2 only ~3 min – i.e. transcription returns to basal i.e transcription immediately after inducer removed immediately • translation of lac mRNA produces translation lac β -galactosidase and other products ∀ β -galactosidase is more stable than -galactosidase the mRNA, so enzymatic activity remains induced for a longer period of time of • Partial diploids used to identify Partial regulatory genes in the lac operon regulatory Lewin Genes VII 2000 Oxford University Press lac OPERON MERODIPLOID lac MERODIPLOID ANALYSIS ANALYSIS • Partial diploids Partial (merodiploids): used to ): determine genetic complementation in bacteria bacteria • E.g. through Hfr E.g. conjugation, additional copy of lac operon copy introduced: introduced: I+ P+ O+ Z+ Y+ A+ I- P+ O+ Z- Y+ A+ Partial diploid cells show that functional repressors work in trans in the absence of lactose, the lac genes are not expressed, and so no significant level of beta-galactosidase is made in these cells (recessive mutation) Partial diploid cells show that operators work only in cis. The lac genes attached to the mutant operator (Oc) continue to express constitutively (all the time) even in the presence of a wild-type operator on another chromosome in the same cell. MUTATIONS IDENTIFYING REGULATORY GENES & SEQUENCES REGULATORY OPERATOR MUTATIONS: OPERATOR OC cis-DOMINANT cis • lac operator: defined by the OC mutation that causes lac lac operon constitutive expression expression • An extra copy of the lac An lac operon cannot repress OC cannot constitutive expression: constitutive I+ P+ O+ Z+ Y+ A+ I+ P+ OC Z+ Y+ A+ • OC operator mutation only operator contro...
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