The Lac Operon
THE LAC OPERON.......................................................................................................................................... 1
THE IMPORTANCE OF MUTATIONS............................................................................................................................2
THE LAC OPERON MODEL.......................................................................................................................................2
What were the Proofs of the Lac operon Model?..........................................................................................2
What is the difference between an Operon and a Regulon?.........................................................................2
Structure:........................................................................................................................................................2
REGULATION OF THE LAC OPERON............................................................................................................................3
Positive regulation:.........................................................................................................................................3
Negative Regulation:......................................................................................................................................3
Catabolite Repression:....................................................................................................................................3
TRANS AND CIS-ACTING CONTROL ELEMENTS.............................................................................................................4
Trans-acting control (via proteins).................................................................................................................4
Cis-acting control elements (DNA Recognition sites).....................................................................................4
INDUCTION OF THE LAC OPERON..............................................................................................................................4
PROOFS........................................................................................................................................................ 4
1. PROOF THAT GENES ARE TRANSCRIBED ON SINGLE MRNA TRANSCRIPT.........................................................................5
Mechanism:....................................................................................................................................................5
2. PROOF THAT ALLOLACTOSE AND NOT LACTOSE IS THE INDUCER...................................................................................5
Recap: Lac Operon Repression & Induction:...................................................................................................5
Experiment 1: Use a lacZ- lacY+ mutant.........................................................................................................6
Experiment 2: Use lacZ- lacY+ mutant............................................................................................................6
3. PROOF THAT LACI ENCODES A REPRESSOR...............................................................................................................6
Results:...........................................................................................................................................................6
4. PROOF OF THE ROLES OF CIS AND TRANS-ACTING ELEMENTS.......................................................................................7
Proof Trans-acting element: Experiment 1.....................................................................................................7
Proof Trans-acting element: Experiment 2.....................................................................................................8
Proof Cis-acting element: Experiment 1.........................................................................................................8
Proof Cis-acting element: Experiment 2.........................................................................................................9
5. PROOF LACI REPRESSOR BINDS TO OPERATOR REGION IN PROMOTOER.........................................................................9
The lac Repressor:...........................................................................................................................................9
Key Features of typical bacterial vector for Protein Production:....................................................................9
LacI Purification Method:.............................................................................................................................10
Gel shift assay:..............................................................................................................................................10
DNAse Foot printing technique:...................................................................................................................10
Findings:.................................................................................................................................................................. 11
6. PROOF THAT LACI BINDS TO INDUCER MOLECULE...................................................................................................12
CATABOLITE REPRESSION............................................................................................................................ 13
Glucose control of cAMP levels:...................................................................................................................13
Control of Adenylate Cyclase Activity:..........................................................................................................13
CAP regulation of Lac operon.......................................................................................................................14
Proof that CAP binds cAMP for positive regulation of lacZ:.........................................................................14
, The Lac Operon
The Importance of Mutations
- Mutations are important tools of genetic analyses.
- Key principles and concepts in genetics have been obtained through analyses of
mutants.
- Mutations allow fundamental biological processes to be dissected so that the overall
biological system can be understood
- A lot of info on lac operon was obtained through study of mutations.
The Lac Operon Model
- Deduced the structure and function of the lac operon using a genetic approach
- Analyzed the interactions of mutations in E. coli that interfered with normal
regulation of the lactose metabolism
- Demonstrated that the operon as the basic unit of transcriptional control in bacteria
o Structural genes (produce structural proteins)
o Regulatory genes (produce regulatory proteins)
o Regulatory DNA sequences (not genes)
What were the Proofs of the Lac operon Model?
- Genes transcribes on single mRNA transcript
- Allolactose and not lactose is the inducer
- lacI encodes a repressor
- Prove roles of cis and trans-acting elements
- LacI binds to Operator region in promoter
- LacI binds to inducer molecule
What is the difference between an Operon and a Regulon?
- Operon:
o Group of genes physically linked to the chromosome under the control of the
same promoter which gives rise to a polycistonic mRNA.
- Regulon:
o Group of genes needed for the same process but located in different parts of
the chromosome and containing their own promoter, but the promoters are
regulated in the same fashion to allow for coordinated expression.
Structure:
- plac = Promoter for structural genes
THE LAC OPERON.......................................................................................................................................... 1
THE IMPORTANCE OF MUTATIONS............................................................................................................................2
THE LAC OPERON MODEL.......................................................................................................................................2
What were the Proofs of the Lac operon Model?..........................................................................................2
What is the difference between an Operon and a Regulon?.........................................................................2
Structure:........................................................................................................................................................2
REGULATION OF THE LAC OPERON............................................................................................................................3
Positive regulation:.........................................................................................................................................3
Negative Regulation:......................................................................................................................................3
Catabolite Repression:....................................................................................................................................3
TRANS AND CIS-ACTING CONTROL ELEMENTS.............................................................................................................4
Trans-acting control (via proteins).................................................................................................................4
Cis-acting control elements (DNA Recognition sites).....................................................................................4
INDUCTION OF THE LAC OPERON..............................................................................................................................4
PROOFS........................................................................................................................................................ 4
1. PROOF THAT GENES ARE TRANSCRIBED ON SINGLE MRNA TRANSCRIPT.........................................................................5
Mechanism:....................................................................................................................................................5
2. PROOF THAT ALLOLACTOSE AND NOT LACTOSE IS THE INDUCER...................................................................................5
Recap: Lac Operon Repression & Induction:...................................................................................................5
Experiment 1: Use a lacZ- lacY+ mutant.........................................................................................................6
Experiment 2: Use lacZ- lacY+ mutant............................................................................................................6
3. PROOF THAT LACI ENCODES A REPRESSOR...............................................................................................................6
Results:...........................................................................................................................................................6
4. PROOF OF THE ROLES OF CIS AND TRANS-ACTING ELEMENTS.......................................................................................7
Proof Trans-acting element: Experiment 1.....................................................................................................7
Proof Trans-acting element: Experiment 2.....................................................................................................8
Proof Cis-acting element: Experiment 1.........................................................................................................8
Proof Cis-acting element: Experiment 2.........................................................................................................9
5. PROOF LACI REPRESSOR BINDS TO OPERATOR REGION IN PROMOTOER.........................................................................9
The lac Repressor:...........................................................................................................................................9
Key Features of typical bacterial vector for Protein Production:....................................................................9
LacI Purification Method:.............................................................................................................................10
Gel shift assay:..............................................................................................................................................10
DNAse Foot printing technique:...................................................................................................................10
Findings:.................................................................................................................................................................. 11
6. PROOF THAT LACI BINDS TO INDUCER MOLECULE...................................................................................................12
CATABOLITE REPRESSION............................................................................................................................ 13
Glucose control of cAMP levels:...................................................................................................................13
Control of Adenylate Cyclase Activity:..........................................................................................................13
CAP regulation of Lac operon.......................................................................................................................14
Proof that CAP binds cAMP for positive regulation of lacZ:.........................................................................14
, The Lac Operon
The Importance of Mutations
- Mutations are important tools of genetic analyses.
- Key principles and concepts in genetics have been obtained through analyses of
mutants.
- Mutations allow fundamental biological processes to be dissected so that the overall
biological system can be understood
- A lot of info on lac operon was obtained through study of mutations.
The Lac Operon Model
- Deduced the structure and function of the lac operon using a genetic approach
- Analyzed the interactions of mutations in E. coli that interfered with normal
regulation of the lactose metabolism
- Demonstrated that the operon as the basic unit of transcriptional control in bacteria
o Structural genes (produce structural proteins)
o Regulatory genes (produce regulatory proteins)
o Regulatory DNA sequences (not genes)
What were the Proofs of the Lac operon Model?
- Genes transcribes on single mRNA transcript
- Allolactose and not lactose is the inducer
- lacI encodes a repressor
- Prove roles of cis and trans-acting elements
- LacI binds to Operator region in promoter
- LacI binds to inducer molecule
What is the difference between an Operon and a Regulon?
- Operon:
o Group of genes physically linked to the chromosome under the control of the
same promoter which gives rise to a polycistonic mRNA.
- Regulon:
o Group of genes needed for the same process but located in different parts of
the chromosome and containing their own promoter, but the promoters are
regulated in the same fashion to allow for coordinated expression.
Structure:
- plac = Promoter for structural genes
