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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......

Bacterial mRNA usually has a shorter lifespan

Posted by star on 2018-10-25 19:15:49
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    An important characteristic of bacterial mRNA is that its lifetime is short compared to other types of bacterial RNA molecules. The half-life of a typical bacterial mRNA molecule is a few minutes. This feature, which may seem terribly wasteful, has an important regulatory function. A cell can turn off the synthesis of a protein that is no longer needed by turning off synthesis of the mRNA that encodes the protein. Soon after, none of that particular mRNA will remain and synthesis of the protein will cease. Of course, this regulation also means that in order to maintain synthesis of a particular protein, the mRNA molecules encoding these proteins must be synthesized continuously. Continuous mRNA synthesis is a small payment by the cell for the ability to regulate the synthesis of specific proteins. This means that in the overall metabolism of a cell, much less ATP is consumed than would be used if synthesis of proteins encoded in the mRNA continued long after the proteins were no longer needed.
    The short lifetime of bacterial mRNA is one criterion used to identify mRNA in bacteria. A common experimental technique to determine whether a particular RNA molecule or class of RNA molecules is mRNA is the pulse-chase experiment. RNA is labeled briefly by growing bacteria in the presence of a radioactive precursor such as [3H]uridine. Then the bacteria are switched to a medium containing no [3H]uridine and a high concentration of nonradioactive urdine and samples are removed at specific times for analyses. The RNA is isolated and different species are separated by gel electrophoresis or centrifugation and detected by their radioactivity. A stable radioactive RNA molecule will be present through many generations, whereas a radioactive mRNA molecule will decrease with a half-life of two to three minutes. One difficulty with this technique is that bacteria contain some long-lived mRNA molecules and these would be misclassified. A b......

Bacterial mRNA

Posted by star on 2018-10-25 19:13:11
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    Our examination of the regulation of mRNA synthesis begins by considering some characteristics of mRNA, the sequence of which is determined by a specific template DNA sequence within the bacterial chromosome. Because transcription proceeds in a 5'→3'direction and transcription and translation occur in the same cellular compartment, the bacterial protein synthetic machinery can start to read the 5'-end of mRNA before the 3'-end is formed. Therefore, a bacterial cell does not have a chance to alter a nascent mRNA molecule before the protein synthetic machinery begins to translate it. The situation is different in eukaryotes. Because transcription occurs in the cell nucleus and translation occurs in the cytoplasm, the eukaryotic cell can convert the primary transcript to mature mRNA in the cell nucleus before the mRNA is required to direct protein synthesis in the cytoplasm.
    Protein synthetic machinery leads the mRNA nucleotide sequence in groups of three bases or codons. Each codon specifies an amino acid or a termination signal. The protein synthetic machinery begins polypeptide synthesis at a start codon located toward the5'_end of the mRNA and continues synthesis in a 5'→3'direction until it encounters a termination codon. The segment of mRNA that codes for a polypeptide chain is called an open reading frame (ORF) because the protein synthetic machinery begins reading the segment at a specific start codon and stops reading it at a specific start codon and stops reads it at a specific termination codon. A DNA segment corresponding to an open reading frame plus the translational start and stop signals for protein synthesis is called a cistron and an mRNA encoding a single polypeptide is called monocistronic mRNA. Although the terms cistron and gene are sometimes used interchangeably to describe bacterial DNA segments that specify polypeptides, the term gene has a broader meaning because it also includes the prom......

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