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The regulation of the lactose system

Posted by star on 2018-11-01 22:48:27
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    Monod and Jacob proposed the operon model explain how the lac system is regulated. The term operon refers to two or more contiguous genes and the genetic elements that regulate their transcription in a coordinate fashion. Promoters had not yet been discovered when Monod and Jacob proposed the operon model but were readily incorporated into the operon model after their discovery. Lac operon mode shows a revised version of the original lac operon model that includes the lac promoter. The five major features of the model are:
    1. The products of the lacZ, lacY and lacA genes are encoded in a single polycistronic lac mRNA molecule.
    2. The promoter for this mRNA molecule is immediately adjacent to the lacO region. Promoter mutations (P-) that are completely incapable of making β-galactosidase, permease, and transacetylase have been isolated. The promoter is located between lacI and lacO.
    3. The operator is a sequence of bases (in the DNA) to which the repressor protein binds.
    4. When the repressor protein is bound to the operator; transcription of lac mRNA cannot take place.
    5. Inducers stimulate lac mRNA synthesis by binding to the repressor. This binding alters the repressor's conformation so it cannot bind to the operator. Thus, in the presence of an inducer the operator is unoccupied and the promoter is available for initiation of mRNA synthesis. This state is called derepression.
    This simple model explains many of the features of the lac system and of other negatively regulated genetic systems. However we will see in a later section that this explanation is incomplete as the lac operon is also subject to positive regulation.



lac mRNA

Posted by star on 2018-10-31 23:13:09
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    Two French investigators, Jacques Monod and Francois Jacob, some-times working together and sometimes with other investigators, performed a series of genetic and biochemical experiments in the late 1950s that helped to elucidate the mechanism that regulates the lac system, They began by isolating constitutive E. coli mutants that make lac mRNA in the presence as well as absence of an inducer. Then they constructed a variety of partial diploid cells containing constitutive mutants and observed the cell's ability to synthesize β-galactosidase.

    These mutations appeared to be of two types, termed lacI and lacOc. The lacI- mutations behave like typical minus mutations in most genes and are recessive (entries 3, 4). Because lac mRNA synthesis is off in a lacl+ cell and on in a lacI- mutant, the lacI gene is apparently a regulatory gene that codes for a product that acts as an inhibitor to keep the lac structural genes turned off. A lacI-mutant lacks the inhibitor and thus is constitutive. A lacl+/lacI- partial diploid has one good copy of the lacI- gene product, so the system is inhibited. Monod and Jacob called the lacI- gene product the Lac repressor. Their original genetic experiments did not indicate whether the Lac repressor is a protein or an RNA molecule.

    This question was answered when an E. coli mutant with a polypeptide chain termination codon inside lacI was isolated and found to synthesize β-galactosidase constitutively. The most likely explanation for the constitutive lactose system is that the mutant strain synthesizes a truncated repressor protein that cannot block transcription of the lac genes. This conclusion was confirmed when the Lac repressor was purified and chara......

Changes of environmental lac in E. coli

Posted by star on 2018-10-30 23:39:45
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    When E. coli with a lac+ genotype is cultured in a lactose-free medium, the intracellular concentrations of β-galactosidase, permease, and transacetylase are exceedingly low-roughly one or two molecules of each protein per bacterium. However, when lactose is added to the growth medium, the concentration of these proteins increases simultaneously to about 105 molecules per cell (or about 1% of the total cellular protein). Furthermore, lactose addition triggers the synthesis of lac mRNA as evidenced by studies in which mRNA, labeled with [32P] phosphate at various times after lactose addition, is hybridized to DNA that carries lac genes.
    Enzymes such as β-galactosidase, lactose permease, and transacetylase are said to be inducible enzymes because their rate of synthesis increases in response to the addition of a small molecule (lactose) to the medium. Other enzymes, called repressible enzymes, exhibit a decreased rate of synthesis in response to the addition of a small molecule in the medium. For instance, the addition of tryptophan to the growth medium causes E. coli to greatly decrease the rate at which it produces enzymes needed for tryptophan synthesis. Still other enzymes, called constitutive enzymes, are synthesized at fixed rates under all growth conditions. Constitutive enzymes usually perform basic cellular "house-keeping" functions needed for normal cell maintenance.
    Lactose is rarely used in experiments to study induction because the β-galactosidase that is synthesized catalyzes the cleavage of lactose. Thus, the lactose concentration continually decreases, which complicates the analysis of many types of experiments (e. g., kinetic experiments). Instead, two sulfur-containing analogs of lactose are used, isopropylthio-galactoside (IPTG) and thiomethylgalactoside (TMG), which are effective inducers without being substrates of β-galactosi......


    In E. coli, two proteins are necessary for the metabolism of lactose. These proteins are the enzyme β-galactosidase, which cleaves lactose to yield galactose and glucose, and a carrier molecule, lactose permease, which is required for the entry of lactose (and other galactosides) into the cell. The existence of two proteins was first shown by a combination of genetic experiments and biochemical analysis.
    First, hundreds of Lac-mutants (unable to use lactose as a carbon source) were isolated. By genetic manipulation, some of these mutations were moved from the E. coli chromosome to an F'lac plasmid (a plasmid carrying the genes for lactose utilization) and then partial diploids having the genotypes F'lac-/ lac+ or F'lac+/lac- were constructed. (The relevant genotype of the plasmid is given to the left of the diagonal line and that of the chromosome to the right.) It was observed that these diploids always produced a Lac+ phenotype, which showed that none of the lac- mutants make an inhibitor that prevents functioning of the lac gene.
    Partial diploids were also constructed in which both the chromosome and the F'lac plasmid were lac-. Using different pairs of lac- mutants. It was found that some pairs were phenotypically Lac+ and some were Lac-. This complementation test showed that all of the mutants initially isolated fell into one of two groups, which were called lacZ and lacY. Mutants in the two groups z and y have the property that the partial diploids F'lacY- lacZ+/lacy+ lacz- and F'lacY+lacZ-/ lacY- lacZ+ have a Lac+ phenotype and the genotypes F'lacY- lacZ+/lacy+ lacz- and F'lacY+ lacZ-/lacy+ lacz- have the......

The role of gene regulation in metabolic activities

Posted by star on 2018-10-29 02:15:34
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    Natural selection improves efficiency. Among the unicellular organisms, any mutation that increases the overall efficiency of cellular metabolism should enable a mutant cell to grow slightly faster than a wild-type organism thus, if enough time is allowed, a mutant cell line will outgrow a wild-type one. For example, in a population of 109 bacteria with a 30. 0-minute doubling time if one bacterium is altered such that it has a 29. 5-minute doubling time, in about 80 days of continued growth, 99. 9% of the population will have a 29. 5-minute doubling time. (In this calculation, it is assumed that the growing culture is repeatedly diluted. Otherwise, unless the volume was greater than that of the earth, the culture would stop growth in a day or so.)
    This time frame may seem very long on the laboratory time scale but it is infinitesimal on the evolutionary scale; thus, it is reasonable on this basis alone that regulated systems, in which efficiency has been improved, should have evolved.
    Bacterial cells increase their efficiency (and therefore their growth rates) by selecting the catabolic pathway for energy production that yields the greatest amount of energy per unit time and synthesizing molecules only as the need arises. Bacteria accomplish both of these objectives by turning on the transcription of specific genes when their products are needed and turning off their transcription when their products are not needed. Actually, there are no known examples of switching a system completely off. When transcription is in the "off" state, there always remains a basal level of gene expression. This basal level often amounts to only one or two transcription events per cell generation and thus very little mRNA synthesis. For convenience, when discussing transcription, we use the term off, but it should be kept in mind that what is meant is very low. We will also see examples in this chapter of systems in w......

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