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LACTOSE OPERON

HISTORY OF LAC OPERON 1900 - It was shown that when lactose is present in the growth medium of yeast, the organisms synthesize enzymes required for lactose metabolism. - When lactose is absent, the enzymes are not manufactured. - Investigators were able to generalize that bacteria also adapt to their environment by producing certain enzymes only when specific chemical substrates are present. 1946 Jacques Monod Joshua Lederberg studied genetic and biochemical evidences involving lactose metabolism Francois Jacob Andre Lwoff Joshua Lederberg first isolated and studied lacmutants -mapping studies established that all three genes are closely linked or contiguous to one another on the bacterial chromosomes, in the order Z-Y-A. Around 1960 Francois Jacob and Jacques Monod proposed a scheme involving negative control called the operon model, where by a group of genes is regulated and expressed together as a unit. Suggested that the repressor normally binds to the DNA sequence of operator region. 1966 Walter Gilbert and Benno Muller Hill reported the isolation of the lac repressor

LACTOSE METABOLISM IN PROKARYOTES REGULATED BY INDUCIBLE SYSTEM Insights were provided in to the way in which gene activity is repressed when lactose is absent, but induced when lactose is available. In the presence of lactose, concentrations of the enzymes responsible for lactose metabolism increase rapidly from a few molecules to thousands per cell. The enzymes responsible for lactose metabolism are thus inducible, and lactose serves as the inducer.

CIS-ACTING SITE AND TRANS-ACTING MOLECULES Cis acting site the location of the regulatory region is almost always upstream of the gene cluster it controls. Trans acting molecules molecules that bind to the cis acting regulatory regions that control transcription of the gene cluster. Binding of a trans acting molecules at a cis acting site can regulate the gene cluster either negatively (by turning off transcription) or positively (by turning on transcription of genes in the cluster).

STRUCTURAL GENES

Structural genes are genes coding for the primary structure of the enzymes. Three structural genes in the lac operon: The lacZ gene encodes , an enzyme whose primary role is to convert lactose to glucose and galactose. The lacY gene specifies the primary structure of permease, an enzyme that facilitates the entry of lactose into the bacterial cell. The lacA gene codes for enzyme transacetylase, the physiological role is still not completely clear, it may involved in the removal of toxic by products of lactose digestion from the cell.

DISCOVERY OF REGULATORY MUTATIONS Gratuitous inducers isopropylthiogalactosidase - They behave like natural inducers, but they do not serve as substrates for enzymes that subsequently synthesized. Constitutive mutants cells bearing this type of mutations produced enzymes regardless of the presence or absence of lactose The studies of the constitutive mutation lacI (repressor gene) mapped the mutation to a site on the bacterial chromosome close to, but distinct from the three structural genes. The lacOC is another set of constitutive mutations producing identical effects to those of lacIis present, which is located in the operator region of the operon.

THE OPERON MODEL: NEGATIVE CONTROL Jacob and Monod argued that lacI gene regulates the transcription of the structural genes by producing a repressor molecule, and that repressor is allosteric. They also suggested that the repressor normally binds to the DNA sequence of the operator region. When it binds, it inhibits the action of RNA polymerase, effectively repressing the transcription of the structural genes. When lactose is present, it binds to the repressor and causes allosteric conformational change. In the absence of the repressor operator interaction, RNA polymerase transcribes structural genes, and the enzymes necessary for lactose metabolism are produced. Because transcription occurs only when the repressor fails to bind to the operator region, regulation is said to be under negative control.

THE CONSTITUTIVE MUTATIONS INTERFERING WITH MOLECULAR INTERACTIONS Both Iand OC constitutive mutations can interfere with the molecular interactions, thus allowing the continuous transcription of the structural genes. In the case of Imutants, the repressor protein is altered or absent and cannot bind to the operator region, so the structural genes are always transcribed. In the case of OC mutant, the nucleotide sequence of the operator DNA is altered and will not bind with a normal repressor molecule, so the result is that the structural genes are always transcribed.

CATABOLITE ACTING PROTEIN (CAP) EXERTS POSITIVE CONTROL CAP is a molecule involved in effectively repressing the expression of the lac operon when glucose is present. This inhibition is known as catabolite repression. It has revealed that RNA polymerase binding is never been efficient unless CAP is also present to facilitate the process. In the absence of glucose and under inducible conditions, CAP exerts positive control by binding to the CAP site, facilitating RNA polymerase binding at the promoter and thus transcription. Therefore, for maximal transcription of structural genes, the repressor must be bound by lactose and CAP must be bound to the CAP binding site.

ROLE OF GLUCOSE IN CAP BINDING CAP binding is dependent on another molecule, cyclic adenosine monophosphate (cAMP). In order to bind to the lac operon promoter, CAP must be bound to cAMP. The level of cAMP is dependent on an enzyme, adenyl cyclase, which catalyzes the conversion of ATP to cAMP. The role of glucose in catabolite repression is to inhibit the activity of adenyl cyclase, causing a decline in the level of cAMP in the cell. Therefore, CAP cannot form the CAP-cAMP complex essential to the positive control of transcription of the lac operon. Activator protein Not bound to DNA Repressor protein Lifted off operator site RNA polymerase Keeps falling off promoter site lac Operon No transcription

Carbohydrates + GLUCOSE + LACTOSE + GLUCOSE - LACTOSE - GLUCOSE - LACTOSE - GLUCOSE + LACTOSE

Not bound to DNA

Bound to operator site

Blocked by the repressor

No transcription

Bound to DNA

Bound to operator site

Blocked by the repressor

No transcription

Bound to DNA

Lifted off operator site

Sits on the promoter site

Transcription

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