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Lac Operon Notes

The lac operon controls the breakdown of lactose in E. coli. It contains the genes lacZ, lacY, and lacI. LacZ encodes the enzyme beta-galactosidase which breaks down lactose, and lacY encodes lactose permease which transports lactose into the cell. LacI encodes the lac repressor which binds to the lac operator and prevents transcription under normal conditions. In the presence of lactose, it binds to the repressor and prevents it from binding to the operator, allowing transcription and expression of the genes. Glucose presence also regulates the system by inhibiting cAMP production, which is needed along with lactose for full induction of the genes.

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36 views

Lac Operon Notes

The lac operon controls the breakdown of lactose in E. coli. It contains the genes lacZ, lacY, and lacI. LacZ encodes the enzyme beta-galactosidase which breaks down lactose, and lacY encodes lactose permease which transports lactose into the cell. LacI encodes the lac repressor which binds to the lac operator and prevents transcription under normal conditions. In the presence of lactose, it binds to the repressor and prevents it from binding to the operator, allowing transcription and expression of the genes. Glucose presence also regulates the system by inhibiting cAMP production, which is needed along with lactose for full induction of the genes.

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kwfengchan
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© © All Rights Reserved
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Download as DOCX, PDF, TXT or read online on Scribd
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Lac Operon

Trans acting factor: external factor (protein) that binds to regulatory DNA sequence

Cis acting sequence: Site where trans acting factor binds.

Trans acting factor stops transcription and gene is inactive

Regulatory region: Contains several protein binding sites – cis acting sequences where trans acting
factors bind

Promoter: RNA poly binds

Activator binding site: Proteins bind to affect freq of transcription

Repressor binding sites: blocks transcription

Positive regulation: Binding of activator protein increases prob of RNA poly binding to the promoter and
therefore increases gene transcription.

Negative regulation: Repressor blocks binding of RNA poly and therefore no transcription occurs.

Mutations that affect gene activity

Mutation that wouldn’t allow RNA poly to bind

Mutation at activator site (loss of function)

Mutation at repressor site (transcription goes on and on)

Lac operon: System where lactose gets broken down.

Beta galactosidase: breaks down lactose disaccharide into glucose and galactose
(E coli uses lactose as energy and carbon source)

Cells grown on glucose cannot break down lactose as there is very little beta g.

When glucose is absent, beta g activity is induced by lactose. Environmental factor control

Lac I sequence: Produces Lac repressor

Lac repressor: binds to Lac O site to stop transcription.

Lac O: Site where repressor binds

Lac I sequence  Lac repressor (binds to) Lac O  RNA poly cannot bind

Lac Y and Lac Z sequence are in the lac operon and are under control of a single lac promoter and are
coordinately expressed. Both proteins produced from a single mRNA that is made whenever promoter is
active. Lac Y and Y are called structural genes because they code for primary structure of proteins. Lac Z
makes beta g and Lac Y makes lactose permease.

Permease brings lactose into cell  Beta g breaks it into simple sugars.
When lactose isn’t present, lac I repressor binds to lac o site and stops transcription and production of
beta g and lac permease

In the presence of lactose, repressor cannot bind to the Lac O site and transcription occurs because
RNA poly can bind. This is because lactose binds on the repressor and makes it misshapen. Therefore it
cannot bind to Lac O site

Binding of a ligand (molecule to protein) causes a change in conformation of the protein and therefore
affects its activity

Lac repressor forms a loop that creates a “barrier” for the poly complex.

Operator site (Lac O) has 2 binding sites, each are a palindromic sequence. The dimers bind and cause
DNA to loop out.

How to identify mutants?

Make cells with one mutant and one wild type copy. If the mutant is in the Lac I sequence, then the wild
type will produce repressor and in the absence of lactose will bind to both the Lac O sites on both
mutant and wild type. Production of lac z and y will be inhibited on both wild type and mutant.
Phenotype would be normal.

If the mutation were in the Lac O site, Lac Z and Y would be produced constitutively in the mutant
because the deferctive operator cannot bind with the repressor.

Cells growing in glucose use glucose. If switched to lactose, they will use lactose via the induction of beta
g activity. If both present, they will preferentially use glucose

Glucose acts as a feedback pathway. The presence of it will stop the activity of beta g.

High [glucose] = low [cAMP]

cAMP binds to CRP site and increases the rate at which transcription occurs

High glucose inhibits adenyl cyclase reaction that produces cAMP from ATP

When [glucose] is low, the rxn is not inhibited, therefore cAMP is made and can bind to CRP site and
therefore there is a high rate of transcription

To get lac activity, you need:


Lactose to remove lac oppressor (lac repressor cannot bind with lactose present)

Low [glu] to allow cAMP production to bond to CRP

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