Thelac. Operon Laboratory Purpose: Learning to analyze an example of gene expression Example: lac operon Type of control: negative inducible
Today’s Laboratory n Work on mutational analysis problem of lac operon; page 3 -24
Lac Operon: Negative Control and Induction
Repressor has two binding sites. Lactose binding site DNA binding site
The promoter needs to be in an ideal conformation for RNA polymerase.
An overview of regulation of the lac operon
Let review a few terms and then look at the lab problem (table) on page 3 -24 important terms of DNA control.
3 important terms of DNA control. n n n Constitutive activity Trans acting Cis acting
Examples of cis and trans acting elements
Cis acting: c mutation O
Trans acting I mutation
Trans acting: s I mutation
Laboratory Exercise
Haploid and Heterozygous Diploid Mutants n n Predict if you expect to obtain: transcription/translation of the lac. Z and lac. Y genes when lactose is absent or present for the E. coli.
Try out your ability to reason through the questions.
PROTEIN PRODUCT Β-galactosidase No Lactose I+P+O+Z+Y+ I-P+O+Z+Y+ I+P-O+Z+Y+ I+P+Oc. Z+Y+ I+P+O+Z-Y-/ I-P+O+Z+Y+ I+P+Oc. Z+Y-/ I+P+O+Z-Y+ I+P+Oc. Z-Y+/ I-P+O+Z+YISP+O+Z+Y-/ I-P+O+Z-Y+ I+P-Oc. Z+Y+/ I+P+O+Z-Y- - Lactose + Permease No lactose - Lactose +
The next slides are a “key” to the mutations.
Experimental tests on the repressor: n I+ cells synthesize full levels of the lac enzymes only in the presence of an inducer n I- mutations NO DNA BINDING ACTIVITY, TRANSCRIPTION OCCURS EVEN IN ABSENCE OF INDUCER! n Is mutation NO LACTOSE BINDING ACTIVITY but has DNA BINDING ACTIVITY. NO TRANSCRIPTION EVEN WITH INDUCER!
Experimental mutations to the repressor I s=inducer binding region mutated Inhibits transcription, even with inducer Lactose binding site I - = DNA binding site mutated, prevents binding, allows transcription DNA binding site
The End.
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