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cAMP · CRP activates more than 100 operons

Posted by star on 2018-11-13 22:03:45
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    Enzymes responsible for the catabolism of many other organic molecules, including galactose, arabinose, sorbitol, and glycerol, are synthesized by inducible operons. Each of these operons cannot be induced if glucose is present. These are called catabolite-sensitive operons. A network of operons that is under the control of a single regulatory protein such as cAMP · CRP is called a modulon.
    A simple genetic experiment shows that cAMP · CRP participates in the regulation of many operons. A single spontaneous mutation in a gene for the catabolism of a sugar such as lactose, galactose, arabinose, or maltose arises with a frequency of roughly 10-6. A double mutation, lac-mal-, would arise at a frequency of 10-12, which for all practical purposes cannot be measured. However double mutants that are phenotypically Lac- Mal- or Gal- Ara- do arise at a measurable frequency. These apparent double mutants are not the result of mutations in the two sugar operons but always turn out to be crp- or cya-. Furthermore, if a Lac- Mal- mutant appears as a result of a single mutation, the protein products of the other catabolite-sensitive operons are also not synthesized. Biochemical experiments with a few of these catabolite-sensitive operons indicate that binding of cAMP · CRP occurs in the promoter region in each of these systems.




    In the absence of cAMP· CRP, the lac promoter is quite weak because its-10 box differs significantly from the consensus sequence. A mutant lac promoter with a -10 box that has the consensus sequence does not re-quire cAMP · CRP for transcription activation. It therefore seems reasonable to propose that interactions between cAMp· CRP complex and RNA polymerase holoenzyme increase the holoenzyme's affinity for the lac promoter. Biochemical and genetic studies support this proposal.
    Thomas Steitz and coworkers determined the crystal structure for the cAMP· CRP complex bound to DNA. CRP consists of two chemically identical polypeptide chains of 209 amino acid residues. Each chain consists of an N-terminal domain and a C-terminal domain, which are connected by a hinge region containing four amino acids. The N-terminal domain consists of a series of antiparallel β-sheets that form a pocket for binding cAMP The C-terminal domain contains a helix-turn-helix motif that binds to DNA. In the absence of cAMP, CRP · DNA interactions are nonspecific and weak. However, the cAMP · CRP complex binds very tightly to a specific DNA sequence designated the activator site (AS). In the lac operon, the center of AS is 61. 5 bp upstream from the transcription start site. Many other bacterial operons are activated by cAMP · CRP (see below). Each of these operons also contains at least one AS. Comparing AS sequences reveals the following 22 bp consensus sequence with twofold symmetry.


5'-AAATGTGATCTAGATCACAT T T-3'
3'-T T TACACTAGATCTAGTGTAAA-5'


    The most highly conserved nucleotides in AS are the two TGTGA motifs. Mutations that alter nucleotides in the TGTGA motifs lead to decreased lac operon transcription. Each CRP subunit binds to half of the AS. The interacti......


    The mechanism by which glucose inhibits β-galactosidase synthesis remained a complete mystery for about 20 years after Monod first observed the phenomenon. Richard S. Makman and Earl W. Sutherland found an important clue to the mystery in 1965 when they observed that the intracellular concentration of 3', 5'-cyclic adenylate or cAMP drops from about 10-4 M to 10-7 M when glucose is added to a growing culture of E.coli.
    Genetic studies confirmed cAMP's involvement in catabolite repression. Two mutant classes were isolated that could not synthesize lac enzymes when cultured in a medium containing lactose but no glucose. Class mutants regained the ability to synthesize lac enzymes when cAMP was added to the growth medium but class ‖ mutants did not.
    Subsequent studies showed that class I mutants have defects in adenylate cyclase, the enzyme that converts ATP to cAMP. The structural gene for adenylate cyclase, cya, maps at minute 85.98. Adenylate cyclase exists in an active form that is phospfborylated, (it contains an attached phosphate group) and an inactive form that is dephosphorylated. Class ‖ mutants have defects in a protein that binds cAMP. This protein, called the cAMP receptor protein (CRP) or the catabolite activator protein (CAP), is encoded by the crp gene, which maps at minute 75. 09. In vitro studies have shown that CRP and cAMP form a complex, denoted cAMP·CRP complex, which is needed to activate the lac system. The cAMP · CRP requirement is independent of the repression system since cya and crp mutants cannot make lac mRNA even if a lacI- or lacOc mutation is present. Thus, cAMP · CRP is a positive regulator or activator, in contrast to the repressor and the lac operon is independently regulated both positively and negatively.
    Based on the information presented above, it seems reasonable to propose that glucose somehow inhibits phospho......

cell combination drug delivery ---New technology for treating AML

Posted by star on 2018-11-07 22:05:41
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    on October 29th ,2018,“Conjugation of haematopoietic stem cells and platelets decorated with anti-PD-1 antibodies augments anti-leukaemia efficacy”was published in Nature Biomedical Engineering. The researchers proposed a "cell combination drug delivery" (CCDD) technology: two different cells are glued together to target drug delivery.It has been verified on animal models that the anti-cancer drug can be delivered to the deep bone marrow to enhance the efficacy of killing cancer cells and significantly inhibit recurrence.
    This technology is currently targeted at acute myeloid leukemia (AML). This is a cancer that develops in the bone marrow. Generally speaking, for AML patients, the usual treatment is chemotherapy, but its effect is not so ideal. Usually because some cancer cells are hidden in the bone marrow, chemotherapy does not "deep into the bone marrow.
    CCDD technology is the first treatment to connect two different cells together. Among them, one type of cell is a platelet which is used to deliver an immunotherapy drug PD-1 antibody. These drugs can find cancer cells and destroy their defense systems. Another type of cell is a hematopoietic stem cell(HSC). HSC are like a signal pointing to the bone marrow. Once stem cells bring cell combinations (ie, stem cells and platelets) to the bone marrow, platelets can be activated, releasing drugs and exerting anticancer effects.
    The researchers injected HSC-platelet-aPD-1 conjugates into these leukemia mouse models.
    After 3 weeks of treatment, the cancer lesions in the mice were effectively reduced. In many control groups, cancer has intensified.In terms of survival rate, nearly 90% of the mice receiving the new treatment lived for more than 80 days.Mice in the control group can only live up to 40 days. This therapy not only treats patients individually, but can also be combined with chemo......

The Lac repressor is a dimer of dimers

Posted by star on 2018-11-07 18:13:24
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    Mitchell Lewis and coworkers obtained the crystal structure for the Lac repressor bound to IPTG or lac DNA in 1996. It shows the structure of one of the four identical Lac repressor subunits. As indicated in the figure, the monomer has four distinct functional units:
    1. N-terminal domain or headpiece (residues 1-45) binds lac operator DNA. A helix-turn-helix motif within the head-piece recognizes and binds appropriate operator DNA. Helix-turn-helix motifs, which are often present in proteins that bind to DNA, consist of two short α-helical segments containing between 7 and 9 residues that are separated by a β-turn (a tight turn involving four amino acid residues). One of the two α-helices, designated the recognition helix, is positioned in the major groove of the DNA so that amino acid residues within this helix make specific contacts with bases in the DNA. The other helix, which is designated the stabilization helix, helps to stabilize the complex.
    2. The hinge region (residues 46-62), which joins the N-terminal headpiece to the core domain, is disordered in the absence of DNA but folds into an α-helix upon binding to operator DNA. Two hinge helices in a Lac repressor dimer associate through van der Waals interactions and the paired hinge helices bind to the center of the lac operator in the minor groove and bend the DNA.
    3. The ligand binding or core domain (residues 62-340) is divided into an N-terminal sub-domain and a C-terminal sub-domain. The two kinds of sub-domain have similar folding patterns, Weak non-covalent interactions between N-terminal sub-domains as well as between C-terminal sub-domains make important contributions to Lac repressor dimer formation. IPTG fits in a pocket between the two sub-domains in each polypeptide.
    4. The tetramerization helix (residues 341-357) associates with corresponding regions of the......

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