Slides-Two-Comp2008_1

Slides-Two-Comp2008_1 - Two Component Regulatory Systems...

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Unformatted text preview: Two Component Regulatory Systems Outline 1.  Overview of 2-component systems – modular 2.  Discovery of 2-component systems – nitrogen sensing in E. coli Response to Nitrogen Limitation •  The levels of the glutamine synthetase protein increase as the concentration of NH4+ decreases. •  Glutamate + ATP + NH4+ via Glut. Syn. Glutamine + ADP How are cells sensing NH4+ limitation and increasing the concentration of Gln Syn? Gene Expression gene transcription translation protein Hypothesis: Transcription of the gene for gln syn is up-regulated under conditions of NH4+ limitation. glnA mRNA levels increase after shift of cells to a medium lacking NH4+ -2 +7 +20 +40 Time in minutes relative to shift to medium lacking NH4+ Reitzer LJ & Magasanik B 1985 PNAS 82:1979-1983 How are glnA mRNA levels changing? •  Hypothesis: •  There is a protein that increases transcription of glnA and this protein is only active or present in N-limited cells. •  If so, we should be able to isolate a mutant strain of E. coli in which the gene encoding this protein is mutated and non-functional. •  Such a mutant should have decreased expression of glnA at in N-limited cells. Mutants that lack glnG or glnL do not induce glnA expression Strain NH4+ WT + - glnL- + - Reitzer LJ & Magasanik B 1985 PNAS 82:1979-1983 glnG- + - What are the function of GlnL and GlnG? •  GlnG has similarity to DNA-binding proteins. GlnL does not look like anything else of known function. •  Hypothesis: GlnG is a transcriptional activator of glnA. •  Experiment: in vitro transcription assay +/- GlnG Purified GlnG does not affect transcription of glnA in vitro core RNA pol. sigma GlnG supercoiled Hunt TP & Magasanik B 1985 PNAS 82:8453-8457 linear What is the function of GlnL? •  Hypothesis: GlnL works in conjuction with GlnG to activate transcription of glnA. •  Experiment: in vitro transcription assay + GlnG and +/- GlnL. Both GlnL and GlnG are necessary to stimulate transcription of glnA in vitro +GlnG [GlnL] 0 10 5 Nifa AJ & Magasanik B 1986 PNAS 83:5909-5913 2 1 Observation •  In vitro transcription assay •  One method – pre-incubate with ATP, GTP, and CTP – then start transcription with UTP •  Second method – no pre-incubation, just add ATP, GTP, CTP, UTP to start transcription. •  Result: GlnL did not stimulate glnA transcription in the second method. •  Hypothesis: Nucleotides are required for GlnL to stimulate transcription through GlnG. First question though, which nucleotide(s) does GlnL require? ATP is necessary for GlnL and GlnG to stimulate transcription of glnA in vitro Time Nifa AJ & Magasanik B 1986 PNAS 83:5909-5913 Role of ATP •  Hypothesis: Phosphate from ATP is transferred to GlnL and then GlnL transfer the phosphate to GlnG to activate this protein. Covalent linkage of phosphate to protein. •  Assay: Incubate GlnL and GlnG with 32P-ATP, run proteins on SDSpolyacrylamide gel, expose to film. Incubation of GlnG with GlnL and ATP leads to phosphorylation of GlnG SDS-PAGE -GlnL autoradiograph Nifa AJ & Magasanik B 1986 PNAS 83:5909-5913 +GlnL ...
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