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Analyse der transportgesteuerten Genregulation anhand des Mlc-PtsG Systems von Escherichia coli

Analyse der transportgesteuerten Genregulation anhand des Mlc-PtsG Systems von Escherichia coli

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SEITZ, Sabine, 2005. Analyse der transportgesteuerten Genregulation anhand des Mlc-PtsG Systems von Escherichia coli [Dissertation]. Konstanz: University of Konstanz

@phdthesis{Seitz2005Analy-8579, title={Analyse der transportgesteuerten Genregulation anhand des Mlc-PtsG Systems von Escherichia coli}, year={2005}, author={Seitz, Sabine}, address={Konstanz}, school={Universität Konstanz} }

2005 Seitz, Sabine application/pdf Analysis of the transport mediated gene regulation in the Mlc-PtsG-system of Escherichia coli 2011-03-24T17:44:50Z Seitz, Sabine deposit-license deu Analyse der transportgesteuerten Genregulation anhand des Mlc-PtsG Systems von Escherichia coli Mlc from Escherichia coli is a global transcriptional regulator that represses the expression of different systems which are needed for transport and utilization of carbohydrates. On the other hand, the Mlc regulated genes are activated by the cAMP/CAP complex. Among the genes regulated by Mlc are whose products are part of PEP dependent phosphotransferase systems (PTS). In addition these PTS-components have regulatory function for themselves. Thus, EIIAGlc influences the enzyme adenylyl cyclase which makes cAMP from ATP. Mlc itself is inactivated by binding to PtsG, the glucose-specific EIICB. Mlc is bound to dephosphorylated PtsG which is found during active transport of glucose. Thus, in the Mlc-PtsG-system the formation of the activator cAMP/CAP and the activity of the repressor Mlc are regulated in dependence of the active transport of glucose via PtsG.<br /><br />In this paper it was shown that the soluble, cytoplasmic B-domain is sufficient for the binding of Mlc to PtsG, also if this domain is expressed as a single peptide independent of the rest of the protein. Derepression of the Mlc-regulated genes is only achieved when the B-domain is fixed to the membrane with a membrane anchor and therefore the repressor Mlc is titrated to the membrane. In this case it is possible to fuse IIBGlc to a membrane protein which has no similarity to the C-domain of PtsG. Besides, it could be shown that Mlc binds to a region of the B-domain that overlaps with the binding side of EIIAGlc and that is in direct proximity of the phosphorylation site of the B-domain, Cys 421. Arg424 of PtsG which is also involved in the stabilization of the phosphate bond is absolutely required for the binding of Mlc.<br /><br />The determination of the molecular weight of Mlc under native conditions showed that the protein exists as a tetramer. Possibly the C-terminal 18 amino acids of Mlc form an amphipatic -helix. The experiments done in this work showed that the helix has an important function in the formation of the correct tertiary or quaternary structure of the protein. Under native conditions a Mlc-variant which is 18 amino acids shorter solely forms a dimer and has lost the ability to bind to PtsG or to DNA.<br /><br />In additional binding tests it was found that besides PtsG also YeeI is bound to Mlc. YeeI is a soluble protein which when overproduced, positively influences the expression of the Mlc-controlled genes. It could be demonstrated that YeeI forms a dimer under native conditions. Performing in vitro binding tests it was impossible to detach the Mlc-PtsG-bond even if an excess of YeeI was added.<br /><br />Likewise a direct protein-protein-interaction between adenylyl cyclase and EIIAGlc could be shown. In contrast to the predominant model that propagates the binding of phosphorylated EIIAGlc, it was dephosphorylated EIIAGlc which was bound. It is unclear if phosphorylated EIIAGlc is bound too. Binding of EIIAGlc to the N-terminal part of the amino terminal catalytic domain of adenylyl cyclase could be detected.<br /><br />A genome wide search for further Mlc-regulated genes was performed with the help of micro arrays. These data were analysed with the help of reporter gene fusions. Thereby some of the false positive results could be excluded, but up to now no further Mlc-regulated genes could be determined. 2011-03-24T17:44:50Z

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