Скачать презентацию Thelac Operon Laboratory Purpose Learning to analyze an Скачать презентацию Thelac Operon Laboratory Purpose Learning to analyze an

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Thelac. Operon Laboratory Purpose: Learning to analyze an example of gene expression Example: lac 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 Today’s Laboratory n Work on mutational analysis problem of lac operon; page 3 -24

Lac Operon: Negative Control and Induction Lac Operon: Negative Control and Induction

Repressor has two binding sites. Lactose binding site DNA binding site Repressor has two binding sites. Lactose binding site DNA binding site

The promoter needs to be in an ideal conformation for RNA polymerase. The promoter needs to be in an ideal conformation for RNA polymerase.

An overview of regulation of the lac operon An overview of regulation of the lac operon

Let review a few terms and then look at the lab problem (table) on 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 3 important terms of DNA control. n n n Constitutive activity Trans acting Cis acting

Examples of cis and trans acting elements Examples of cis and trans acting elements

Cis acting: c mutation O Cis acting: c mutation O

Trans acting I mutation Trans acting I mutation

Trans acting: s I mutation Trans acting: s I mutation

Laboratory Exercise Laboratory Exercise

Haploid and Heterozygous Diploid Mutants n n Predict if you expect to obtain: transcription/translation 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. 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-/ 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. The next slides are a “key” to the mutations.

Experimental tests on the repressor: n I+ cells synthesize full levels of the lac 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 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. The End.