The principle of gene control Francois Jacob and Jacques Monod The principle of gene control

Francois Jacob and Jacques Monod--principles of gene control|(1961) F.Jacob and J.Monod publish “Genetic regulatory mechanisms in the synthesis of proteins.” A paper in which the theory of the operon is developed. They proposed their now-classical operon model in1961. The core of the model was that the level of proteins in cells was controlled at a genetic level. The theory also predicted the existence of mRNA-an unstable intermediate between the genome and the expressed protein. Before this work the prevailing model was called the instruction hypothesis that stated that all proteins were present in a cell, but that in the absence of an inducer they were not properly folded and were inactive. Jacob and Monod were awarded the Nobel Prize in 1965 for their work on characterising genes involved in lactose utilization

-The order in the lac operon : promoter-operator-lacZ-lacY-lacA,and the regulatory gene lacI is located close to the structural genes, just upstream of the promoter. -The lacI gene has its own promoter and terminator. -The promoter for the lacI is a weak promoter, so relaticely fedw repressor molecules are present in the cell. -The lac repressor protein encoded by lacI is made contitutively but its ability to bind to the operator is affected by the presence of inducer.

The lacI gene encodes the lac repressor. The lac repressor is a sequence-specific DNA-binding protein that Negatively regulates expression of the lac operon structural genes. In genetic terminology: The lac repressor is a trans-acting element trans = across The operator is a cis-acting element cis=near

negative control -RNA polymerase molecules bind to the repressor gene(lacI+) promoter and transcibe the lacI gene -translation produces a polypeptides consisting of 360 amion acids. Four of these polypeptide associate together to form the functional repressor protein. -the functional repressor binds to the operator(lacO+). Whin the repressor is bound to the operator, RNA polmerase can bind to the operon’s promoter but is blocked from initiating transcription of the protein-coding genes. The lac operon is said to be under negative control since the binding of the repressor at the operator site blocks transciption of the structural genes.

The lactose (lac) operon of Escherichia coli -negative regulation 1. + Inducer (lactose/ allolactose) Induced Repressor cannot bind p p t t lacI lacZ lacY lacA o transcripts Repressor -galactosidase Permease Acetylase 2. No Inducer Repressed p Repressor binds to laco t lacI lacZ lacY lacA o transcript

allosteric shift -when wild –type E.coli grows in the presence of lactose as the sole carbon source, some of the lactose transported into the cell is converted by existing molecules of b -galactosidase into a metabolite of lactose called allolactose. - The lac repressor protein also has a recongnition site for allolactose . -when allolactose binds to the repressor, it changes the shape of the repressor—this is called an allosteric shift.

Repressor-inducer binding is in equilibrium In the absence of lactose there are only 1-5 molecules of each of the lacZYA proteins in the cell. Following induction, up to 5000 molecules of b-galactosidase can accumulate within minutes. As lactose is cleaved and used as a carbon source for growth the lactose levels drop. Eventually free repressor molecules will accumulate once more and the lac operon will be switched off.

Jacob and Monod used Genetic Analysis of partial diploids to unravel the role of genes in the lac operon. Mutations were isolated at four loci: The genes lacZ, lacY, lacI and in the operator lacO. All loci were shown to be tightly linked on the E.coli chromosome by genetic linkage analysis. Each mutant had unique properties and behaved differently. Analysis was achieved using Hfr strains to create partial diploids where the new strain had two copies of the lac region.

Important mutation on negative control Different gene mutation will have different effects on the negative control.They will affect the transcription of protein-coding genes(lacZ, lacY and lacA).And thus affect the on and off of the genes. The most important gene mutations in negative control are: ---the lacO gene mutation(lacOc) ---the lacI gene mutation(lacI-)

F’ lacO+ lacZ - lacY+ lacOc lacZ+ lacY- (both gene sets have a normal promoter, and the lacA gene is omitted because it is not important to our discussion) Results: b -galactosidase is synthesized in the absence of inducer, but permease is not. Only when lactose is added to the culture and the alllolactose inducer is produced does permease syntheis occur.

Explanations:the base pair alterations of the operator DNA sequence make it unrecognizable by the repressor protein. Since the repressor cannot bind, the structural genes physically linked to the laOc mutation become constitutively expressed.

Effects of lacI gene mutations (a) the effecst of lacI- mutation on lac operon expression in a haploid (1)the lacI mutations map within the repressor structural gene and result in amino acid changes in the repressor. (2)the repressor’s shape is changed, and it cannot now recognize and bind to the operator. (3)As a consequence, transcription cannot be prevented, even in the absence oflactose, and there is constitutive expression of the lac operon.

(b) the effects of lacI- mutation on lac operon expression in diploid The dominance of the lacI+ (wild-type) gene over lacI- mutants is illustrated for the partial diploid describe eariler, lacI+ lacO+ lacZ - lacY+ lacI - lacO+ lacZ+ lacY- (both gene sets have normal operators and normal promoters, and the lacA gene is omitted because it is not important to our discussion) Results: In the absence of inducer,no b -galactosidaseor permease was produce;both were synthesized in the presence of inducer. The expression of both genes was inducible

Explanations: In the absence of the inducer The defective lacI repressor is unable to bind to either normal operator (lacO+) in the cell. But sufficient normal repressors, produced from the lacI+ gene, are present which bind to the two operators and block transcription of both operons. (2) In the presence of the inducer The wild-type repressors are inactivated, so both operons are transcribed. One produces a defective b -galactosidase and a normal permease, while the other produces a normal b -galactosidase and a defective permease; between them, active b -galactosidase and permease are produced. Thus, in lacI+/lacI- partial diploids, both operons present in the cell are under inducible control.

Other classes of lacI gene mutation ---lacIs (superrepressor) mutatants ---lacI-d (dominance) mutants ---lacIQ (quantity) and lacISQ (superquantity) mutants

Other classes of lacI gene mutation lacIs (superrepressor) mutatants In partial diploids with a lacI+/ lacIs genotype, the lacIs allele is trans- dominant, affecting both operon copies.The lacIs mutants, shows no production of lac enzymes in the presence or absence of lactose. Explanations: (1) The mutant repressor gene produces a superrepressor protein that can bind to the operator, but cannnot recognize the inducer allolactose. (2)The mutant superrepressor bind to the operators regardless of wherether the inducer molecule is present or absent, and transcription of the operons can never occur even in the presence of the inducer. (3)The presence of normal repressors in the cell has no effect on this situation, since once a lacIs repressor is on the operator, the repressor cannot be induced to fall off.

Functions of the repressor protein To conclude, specifically, the repressor is involved in three recognition interactions, any one of which can be affected by mutation: i)binding of the repressor to the operator region ii)binding of the inducer to the repressor iii)binding of individual repressor polypeptides to each other to form the active repressor tetramer.

Further investigation –positive control of the lac operon -Several years after Jacob and Monod proposed their operon model, researchers also found a positive control system that regulates the lac operon other than the negative control -The system function to turn on the expression of the operon and ensure that the lac operon will be expressed only if lactose is the sole carbon source but not if glucose is present as well.

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