What are the components of two-component systems?
- Histidine kinase (HK): receives a signal
- Response regulator (RR): receives signal from HK and transmits it to target
Two-components systems contain proteins of 3 types. What are the 3 types?
- Histidine kinase (HK)
- Response regulator (RR)
- Phosphatase: inactivates the RR
- May be the HK, RR, or separate protein
What are the 2 domains of histidine kinases?
- Input domain
- Transmitter domain
What are the 2 domains of response regulators?
- Receiver domain
- Output domain
What does the phosphatase in a two-component system do?
Dephosphorylates the RR, returning it to the nonstimulated state, where it once again can respond to the signal
What are the steps of signal transduction in two-component systems?
- A transmembrane histidine kinase (HK) is activated by a signal at its N-terminal domain
- The activated protein autophosphorylates in the C-terminal domain
- The response regulator (RR) binds to the C-terminal end of the histidine kinase and the phosphoryl group is transferred from the HK to the RR, thus activating the latter
- The activated RR leaves the HK and stimulates its target
What defines histidine kinases and response regulators?
- HK defined by conserved sequence of about 200 amino acids at the C-terminal end
- RR defined by conserved amino-terminal domain of about 100 amino acids
What two-component systems regulate the genes that respond to anaerobiosis?
- Arc system
- FNR system
- Nar system
Use the figure to describe the metabolic changes E. coli undergoes during anaerobiosis.
A. Aerobic conditions
- Oxidative TCA cycle feed electron into SDH and NADH dehydrogenase
B. Anaerobic conditions
- TCA pathway is noncyclic and reductive because α-ketoglutarate dehydrogenase activity is absent/diminished
What are the histidine kinase and response regulator proteins in the Arc system?
- HK: ArcB
- RR: ArcA
What is the Arc system responsible for?
- Anaerobic repression of genes for:
- TCA cycle enzymes
- Glyoxylate cycle enzymes
- Dehydrogenases for aerobic growth
- Fatty acid oxidation enzymes
- Cytochrome o oxidase - Anaerobic induction of the gene for pyruvate formate-lyase
- Induction in low oxygen of the genes for cytochome d oxidase and cobalamin synthesis
All of the following regulate gene expression by oxygen or
nitrate. Which one is not a two-component system?
- Arc system
- FNR system
- Nar system
FNR system
What types of genes does the FNR system activate? Which genes does it repress?
- Positive regulator of transcription for many genes expressed only during anaerobic growth
- Fumarate reductase
- Nitrate reductase
- Formate dehydrogenase-N
- Aspartase
- Anaerobic fumarase B
- Glycerol-3-phosphate dehydrogenase - Repressor for certain genes expressed only during aerobic growth
What do mutations in the fnr gene result in?
Result in an inability to grow anaerobically on fumarte or nitrate as electron acceptors
How does E. coli regulate the activity of FNR?
Iron-sulfur cluster in FNR
- Active FNR: homodimer of an iron-sulfur protein with an oxygen labile [4Fe-4S] cluster in each monomer
- Upon exposure to oxygen: [4Fe-4S] cluster is oxidized and can even be lost from the protein
- Some of the FNR loses its iron clusters and becomes an apoprotein
- Both protein with oxidized cluster and apoprotein are inactive
- Bind with low affinity to DNA and don’t stimulate transcription
What are characteristics of FNR binding sites?
- Have only a partial match to the consensus sequence
- Located at variable positions within promoter regions
- Directing whether FNR has either a positive or negative effect on
transcription
Where are FNR binding sites located at repressed vs. activated promoters?
- Repressed: locations range from upstream of the 235 hexamer (which binds region 4.2 of RNA polymerase s70), to overlapping the transcription start site (TSS; +1)
- Activated: center of the binding site is 41.5 nt upstream of the TSS
Under anaerobic conditions: When E. coli is given a choice of electron acceptors such as nitrate, nitrite, or
fumarate, what will it choose? Why?
Nitrate
- Has a more positive redox potential (closest to oxygen out of all of those)
- Induces transcription of genes resulting in synthesis of a membrane-bound nitrate reductase
- Represses transcription of genes encoding other reductases
Briefly summarize the path of nitrate reduction.
- Nitrate (NO3-) first reduced to nitrite (NO2-) which is then reduced to ammonia (NH4+)
- 8 electrons are required: 2 to reduced nitrate, 6 to reduce ammonia
- Part of the ammonia is assimilated in to cellular compounds, part is excreted into the medium
What enzymes are involved in nitrate reduction? What genes encode them?
- Nitrate reductase (membrane)
- narG operon (narGHJI) - Nitrite reductase (cytoplasmic)
- nirB operon (nirBDC)
Nar system: What is the purpose of the cytoplasmic nitrite reductase?
Prevent the accumulation of toxic nitrite in the cytoplasm
Give an overview of gene regulation of the Nar system.
- In the presence of excess nitrate: preferential synthesis of
enzymes that use nitrate as an electron acceptor (i.e., nitrate reductase) - In the presence of excess nitrite: preferential synthesis of enzymes that use nitrite as an electron acceptor (i.e. respiratory nitrite reductase, cytochrome c522)
What are the histidine kinase and response regulator proteins in the Nar system?
- HK: NarQ and NarX
- RR: NarL and NarP
Explain the roles of NarX, NarQ, NarL, and NarP in the Nar system?
- NarX and NarQ can autophosphorylate (using ATP as the phosphoryl donor) and transfer the phosphoryl group to NarL
- NarX and NarQ can also negatively regulate the NarL protein by dephosphorylating NarL-P
