Molecular biology homework problem on lac operons! please help! Lac Operon Regul

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Molecular biology homework problem on lac operons! please help!

Lac Operon Regulation: The Genes: Lac operon: 3 ORFs lacZ, lacY and lacA, which encode the proteins beta-galactosidase, lactose permease, transacetylase. Betá-galactosidase catalyzes the breakdown of lactose into glucose + galactose. Lactose permease transports lactose from outside to inside the cell. The function of transacetylase is not yet known Laci => 1 ORF that encodes the lac repressor protein Lac repressor binds the cis-element called the lac operator sequence (aka lacO) in the absence of lactose only. The lac repressor also has a binding pocket for lactose, and if that pocket has lactose bound, the protein has a slightly altered structure that does not let it bind DNA. CRP 1 ORF that encode the catabolite gene activator protein (CAP). CAP binds the cis-element called the CAP binding sequence (aka CBS) only if it is also bound to cyclic AMP (CAMP). If CAMP levels are low, the protein has a slightly altered structure that does not let it bind DNA. CAP can interact with RNA polymerase/ sigma factor to efficiently recruit them to a core promoter. The Promoters: The lac operon core promoter is very weak, which means that it cannot efficiently recruit RNA polymerase sigma factor. In addition to the core promoter, there is a lacO site just downstream, and a CBS site just upstream. The promoter is highly regulated The Lacl core promoter is strong. which means that it alone can efficiently recruit RNA polymerase sigma factor. The promoter is constitutive, which means that it is "on" under most conditions. The Crp core promoter is strong, which means that it alone can efficiently recruit RNA polymerase sigma factor, The promoter is constitutive, which means that it is "on" under most conditions The Signals: Lactose levels, which are determine by the amount of lactose in the media (food source) CAMP levels. However, CAMP levels are inversely related to glucose levels in the media through the enzyme adenylate cyclase (AC), which converts ATP to cAMP when activated through complicated signal cascade involving a kinase, the glucose transporter, etc. For simplicity, we can say that when glucose evels are high. Aiso CAMP levels are low. When glucose levels are low. AC is active, so CAMP levels are high The Assay: x-gal is a lactose analog. that when cleaved by beta-galactosidase becomes blue, E. coli with an active beta-galactosidase will form blue colonies when grown on X-gal. If beta-galactosidase is not present or not functional, the E. coli colonies will be white. Note: X-gal does not bind the lac repressor proteirn CBS lacO core promoter I gene ] open reading frame

Explanation / Answer

Ans: In overall, Lac operon having three regulatory sequences like Lac i, Lac p and Lac o. Three structural genes Lac Z, LacY and Lac A.

Lac operon present in bacterial cells mainly in E.Coli. For E.Coli main bacterial source is carbohydrates especially Glucose. If Glucose present in the medium, E.Coli not depend on Lac operon and directly utilized the Glucose as a carbon source. If medium contains Lactose as a carbon source, then Lac operon activated then convert the lactose into galactose and Glucose. This Glucose is used as a carbon source.

In presence of X-Gal, B- galactosidase (encoded by Lac Z) enzyme reacts with X-gal (Substrate) and gives a blue colored product. So E.Coli colonies appeared as Blue color. If there is no Lac operon activation then colonies have appeared as white.

Note: If there is no carbon source either glucose/lactose, there is no proper growth or no colonies (NCs). Here we neglecting this concept. and both -Glucose/-Lactose considered as operon inactive state.

Mutations

Operon state

+Glucose/ -Lactose

+Glucose/+Lactose

-Glucose/-Lactose

-Glucose/+ Lactose

Operon status

Inactive

Active

White

Blue

Disables Lac O Sequence

No binding of the repressor, lac Operon active

Blue

Blue (NCs)

Blue

Disables CAP binding sequence

Operon

Inactive

white

white (NCs)

Disables Lac repressor DNA binding

Operon Active

Light Blue

blue (NCs)

Blue

Disables lac repressor lactose binding

Operon Inactive

White

White (NCs)

Disables CAP DNA binding

Operon Activity depends on cAMP

White

White (NCs)

Blue

Disables CAP cAMP DNA binding

Operon inactive

White

White (NCs)

Disables Lac I promoter

Operon inactive

White

White (NCs)

Disables Crp Promoter

Operon inactive

White

White (NCs)

Moves Lac O upstream of CBS

Operon Activated

Light blue

Light blue (NCs)

Blue

Disables adenylate cyclase

Operon depends upon glucose

White

white (NCs)

Blue

Complementary Experiment

Disables Lac repressor DNA binding: Add an extra copy of WT lacI

Operon inactive

White

White (NCs)

Disables Lac repressor DNA binding: Add an extra copy of WT Lac operon

Operon activity depends upon C- source

Disables Lac O Sequence: Add an extra copy of WT lacI

Disables Lac O Sequence: Add an extra copy of Lac operon

Disables lac repressor lactose binding: Add an extra copy of WT lacI

Operon partially active

Disables lac repressor lactose binding: Add an extra copy of WT lac

Mutations

Operon state

+Glucose/ -Lactose

+Glucose/+Lactose

-Glucose/-Lactose

-Glucose/+ Lactose

Operon status

Inactive

Active

White

Blue

Disables Lac O Sequence

No binding of the repressor, lac Operon active

Blue

Blue (NCs)

Blue

Disables CAP binding sequence

Operon

Inactive

white

white (NCs)

Disables Lac repressor DNA binding

Operon Active

Light Blue

blue (NCs)

Blue

Disables lac repressor lactose binding

Operon Inactive

White

White (NCs)

Disables CAP DNA binding

Operon Activity depends on cAMP

White

White (NCs)

Blue

Disables CAP cAMP DNA binding

Operon inactive

White

White (NCs)

Disables Lac I promoter

Operon inactive

White

White (NCs)

Disables Crp Promoter

Operon inactive

White

White (NCs)

Moves Lac O upstream of CBS

Operon Activated

Light blue

Light blue (NCs)

Blue

Disables adenylate cyclase

Operon depends upon glucose

White

white (NCs)

Blue

Complementary Experiment

Disables Lac repressor DNA binding: Add an extra copy of WT lacI

Operon inactive

White

White (NCs)

Disables Lac repressor DNA binding: Add an extra copy of WT Lac operon

Operon activity depends upon C- source

White White White (NCs) Blue

Disables Lac O Sequence: Add an extra copy of WT lacI

If added copy within the old operon, Lac operon functional White White White (NCs) Blue

Disables Lac O Sequence: Add an extra copy of Lac operon

Operon is active in old one and the new one is functional regulation White White Light blue/ White (NCs) Blue

Disables lac repressor lactose binding: Add an extra copy of WT lacI

Operon partially active

White White White/ light blue (NCs) Light blue

Disables lac repressor lactose binding: Add an extra copy of WT lac

Old operon is an Inactive and new copy is functional regulation. Light blue Light blue light blue (NCs) Blue

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Molecular biology homework problem on lac operons! please help! Lac Operon Regul

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