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Using systems biology to learn how halo respond to their environment bR converts light energy into chemical energy for making ATP LIGHT ATP ATP ATP Halobacterium cell ATP ATP Bacteriorhodopsin (bR) Note: other proteins are required for this process, but we will simplify and focus on bR Bacteriorhodopsin (bR) is made from a protein called bop and the molecule retinal Bop + retinal = bacteriorhodopsin (bR) Cell membRane Retinal Bop protein Bacteriorhodopsin (bR) Halo change the expression of bR in response to the amount of light in their environment LIGHT PRESENT LIGHT ABSENT (DARK) LIGHT Halobacterium cell When there is more light, halo respond by making more bR LIGHT Halobacterium cell When there is less light, halo make less bR Halo change the expression of bR in response to the amount of light in their environment The amount of bR increases when there is more light. + Light bR Note that the size of nodes in this diagram relates to the amount of light and bR, not the actual size. In other words, the size of bR does not change; a larger node for bR indicates a larger amount of bR. OUR QUESTION: How do Halobacterium cells control the amount of bR expressed in response to light? What is the gene and protein network that regulates the expression of bR? Metabolic data group GG-PP phytoene lycopene beta-carotene retinal bop bacteriorhodopsin (bR) GG-PP phytoene lycopene beta-carotene retinal bop bacteriorhodopsin (bR) Question: How would increasing the amount of GG-PP affect the amount of bacteriorhodopsin (bR)? GG-PP phytoene lycopene beta-carotene retinal bop bacteriorhodopsin (bR) Question: How would increasing the amount of GG-PP affect the amount of bacteriorhodopsin (bR)? GG-PP phytoene lycopene beta-carotene retinal bop bacteriorhodopsin (bR) Question: If the enzyme that converts phyotene to lycopene were missing, how would the amount of bacteriorhodopsin (bR) be affected? GG-PP phytoene lycopene beta-carotene retinal bop bacteriorhodopsin (bR) Question: If the enzyme that converts phyotene to lycopene were missing, how would the amount of bacteriorhodopsin (bR) be affected? Homology GG-PP phytoene lycopene CrtB1 beta-carotene retinal CrtY brp bop bacteriorhodopsin (bR) bat Question: Which enzymes are part of the bacteriorhodopsin network? What other genes are involved in the system? GG-PP phytoene lycopene CrtB1 beta-carotene retinal CrtY brp bop bacteriorhodopsin (bR) bat LIGHT Question: Which protein changes in response to light and affects the expression of other genes? Microarray gene expression GG-PP phytoene lycopene CrtB1 beta-carotene retinal CrtY brp bop bacteriorhodopsin (bR) bat To simplify, focus on the genes and gene products (proteins). Then we’ll see how these affect the metabolites and bacteriorhodopsin (bR). CrtB1 CrtY brp bop bat To simplify, focus on the genes and gene products (proteins). Then we’ll see how these affect the metabolites and bacteriorhodopsin (bR). CrtB1 CrtY brp bop bat Question: Which genes does bat affect? CrtB1 CrtY brp bop bat Question: What happens to the expression of the genes when bat is overexpressed? CrtB1 CrtY brp bop bat Question: What happens to the expression of the genes when bat is overexpressed? CrtB1 CrtY brp bop bat Question: What happens to the expression of the genes when bat is knocked out? CrtB1 CrtY brp bop bat Question: What happens to the expression of the genes when bat is knocked out? CrtY CrtB1 brp bop bat Question: What happens to the expression of the genes when bat is knocked out? CrtY CrtB1 brp bop bat Knocked out bat CrtB1 CrtY brp bop bat Overexpressed bat GG-PP phytoene lycopene CrtB1 beta-carotene retinal CrtY brp bop bacteriorhodopsin (bR) bat Question: Bat affects the amount of proteins in the bR network. How does this affect the amount of bacteriorhodopsin produced? phytoene GG-PP lycopene beta-carotene retinal CrtY CrtB1 brp bop bacteriorhodopsin (bR) bat Knocked out bat GG-PP phytoene lycopene CrtB1 beta-carotene CrtY retinal brp bop bacteriorhodopsin (bR) bat Overexpressed bat phytoene GG-PP lycopene beta-carotene retinal CrtY CrtB1 brp bop Knocked out bat bacteriorhodopsin (bR) bat GG-PP phytoene lycopene CrtB1 beta-carotene CrtY Overexpressed bat retinal brp bop bacteriorhodopsin (bR) bat OUR QUESTION: How do Halobacterium cells control the amount of bR expressed in response to light? What is the gene and protein network that regulates the expression of bR? Our hypothesis – Light Present GG-PP phytoene lycopene CrtB1 beta-carotene retinal CrtY brp bop bacteriorhodopsin (bR) bat LIGHT ADP + P ATP only when light is present Our hypothesis – Light Absent GG-PP phytoene lycopene CrtB1 beta-carotene retinal CrtY brp bop bacteriorhodopsin (bR) bat LIGHT ADP + P ATP only when light is present Known Network – bR Production GG-PP phytoene lycopene CrtB1 beta-carotene retinal CrtY + brp bop + + bacteriorhodopsin (bR) bat LIGHT ADP + P ATP only when light is present Where do the other energy pathways fit (e.g. fermentation)? GG-PP phytoene lycopene beta-carotene CrtB1 Arginine retinal CrtY brp YhdG bop Arginine (outside of cell) (inside cell) bat bacteriorhodopsin (bR) LIGHT ADP + P ATP only when light is present ArgH L-arginosuccinate ArgG arginine ArcA ornithine carbamate CO 2 ArcB ArcC citrulline carbamoyl-PO4 ADP + P ATP NH3 CrtB1 CrtY brp [+] [+] YhdG bop [-] [+] bat [-] [-] [-] [-] [-] ArgH ArcA ArgG ArcB ArcC GG-PP phytoene lycopene beta-carotene CrtB1 Arginine retinal CrtY brp YhdG bop Arginine (outside of cell) (inside cell) bat bacteriorhodopsin (bR) LIGHT ADP + P ArgH L-arginosuccinate ArgG arginine ArcA ornithine carbamate ATP CO 2 ArcB ArcC citrulline carbamoyl-PO4 ADP + P ATP NH3 GG-PP phytoene lycopene beta-carotene CrtB1 Arginine brp CrtY YhdG bop Arginine (outside of cell) retinal (inside cell) bat bacteriorhodopsin (bR) LIGHT ADP + P ATP ArgH L-arginosuccinate arginine ArcA ornithine carbamate ArcB ATP ArcC ArgG CO 2 citrulline carbamoyl-PO4 ADP + P NH3 phytoene GG-PP lycopene beta-carotene CrtY CrtB1 Arginine retinal brp YhdG bop Arginine (outside of cell) (inside cell) bat bacteriorhodopsin (bR) LIGHT ADP + P ArgH L-arginosuccinate ArgG arginine ArcA ornithine carbamate ATP CO 2 ArcB ArcC citrulline carbamoyl-PO4 ADP + P ATP NH3 GG-PP phytoene lycopene beta-carotene CrtB1 Arginine brp CrtY YhdG bop Arginine (outside of cell) retinal (inside cell) bat bacteriorhodopsin (bR) LIGHT ADP + P ATP ArgH L-arginosuccinate arginine ArcA ornithine carbamate ArcB ATP ArcC ArgG CO 2 citrulline carbamoyl-PO4 ADP + P NH3 phytoene GG-PP lycopene beta-carotene CrtY CrtB1 Arginine retinal brp YhdG bop Arginine (outside of cell) (inside cell) bat bacteriorhodopsin (bR) LIGHT ADP + P ArgH L-arginosuccinate ArgG arginine ArcA ornithine carbamate ATP CO 2 ArcB ArcC citrulline carbamoyl-PO4 ADP + P ATP NH3 GG-PP phytoene lycopene beta-carotene CrtB1 Arginine brp CrtY YhdG bop Arginine (outside of cell) retinal (inside cell) bat bacteriorhodopsin (bR) LIGHT ADP + P ATP ArgH L-arginosuccinate arginine ArcA ornithine carbamate ArcB ATP ArcC ArgG CO 2 citrulline carbamoyl-PO4 ADP + P NH3 phytoene GG-PP lycopene beta-carotene CrtY CrtB1 Arginine retinal brp YhdG bop Arginine (outside of cell) (inside cell) bat bacteriorhodopsin (bR) LIGHT ADP + P ArgH L-arginosuccinate ArgG arginine ArcA ornithine carbamate ATP CO 2 ArcB ArcC citrulline carbamoyl-PO4 ADP + P ATP NH3 GG-PP phytoene lycopene beta-carotene CrtB1 Arginine brp CrtY YhdG bop Arginine (outside of cell) retinal (inside cell) bat bacteriorhodopsin (bR) LIGHT ADP + P ATP ArgH L-arginosuccinate arginine ArcA ornithine carbamate ArcB ATP ArcC ArgG CO 2 citrulline carbamoyl-PO4 ADP + P NH3 phytoene GG-PP lycopene beta-carotene CrtY CrtB1 Arginine retinal brp YhdG bop Arginine (outside of cell) (inside cell) bat bacteriorhodopsin (bR) LIGHT ADP + P ArgH L-arginosuccinate ArgG arginine ArcA ornithine carbamate ATP CO 2 ArcB ArcC citrulline carbamoyl-PO4 ADP + P ATP NH3 GG-PP phytoene lycopene beta-carotene CrtB1 Arginine brp CrtY YhdG bop Arginine (outside of cell) retinal (inside cell) bat bacteriorhodopsin (bR) LIGHT ADP + P ATP ArgH L-arginosuccinate arginine ArcA ornithine carbamate ArcB ATP ArcC ArgG CO 2 citrulline carbamoyl-PO4 ADP + P NH3 phytoene GG-PP lycopene beta-carotene CrtY CrtB1 Arginine retinal brp YhdG bop Arginine (outside of cell) (inside cell) bat bacteriorhodopsin (bR) LIGHT ADP + P ArgH L-arginosuccinate ArgG arginine ArcA ornithine carbamate ATP CO 2 ArcB ArcC citrulline carbamoyl-PO4 ADP + P ATP NH3 GG-PP phytoene lycopene beta-carotene CrtB1 Arginine brp CrtY YhdG bop Arginine (outside of cell) retinal (inside cell) bat bacteriorhodopsin (bR) LIGHT ADP + P ATP ArgH L-arginosuccinate arginine ArcA ornithine carbamate ArcB ATP ArcC ArgG CO 2 citrulline carbamoyl-PO4 ADP + P NH3 phytoene GG-PP lycopene beta-carotene CrtY CrtB1 Arginine retinal brp YhdG bop Arginine (outside of cell) (inside cell) bat bacteriorhodopsin (bR) LIGHT ADP + P ArgH L-arginosuccinate ArgG arginine ArcA ornithine carbamate ATP CO 2 ArcB ArcC citrulline carbamoyl-PO4 ADP + P ATP NH3 GG-PP phytoene lycopene beta-carotene CrtB1 Arginine brp CrtY YhdG bop Arginine (outside of cell) retinal (inside cell) bat bacteriorhodopsin (bR) LIGHT ADP + P ATP ArgH L-arginosuccinate arginine ArcA ornithine carbamate ArcB ATP ArcC ArgG CO 2 citrulline carbamoyl-PO4 ADP + P NH3 phytoene GG-PP lycopene beta-carotene CrtY CrtB1 Arginine retinal brp YhdG bop Arginine (outside of cell) (inside cell) bat bacteriorhodopsin (bR) LIGHT ADP + P ArgH L-arginosuccinate ArgG arginine ArcA ornithine carbamate ATP CO 2 ArcB ArcC citrulline carbamoyl-PO4 ADP + P ATP NH3 GG-PP phytoene lycopene beta-carotene CrtB1 Arginine brp CrtY YhdG bop Arginine (outside of cell) retinal (inside cell) bat bacteriorhodopsin (bR) LIGHT ADP + P ATP ArgH L-arginosuccinate arginine ArcA ornithine carbamate ArcB ATP ArcC ArgG CO 2 citrulline carbamoyl-PO4 ADP + P NH3 phytoene GG-PP lycopene beta-carotene CrtY CrtB1 Arginine retinal brp YhdG bop Arginine (outside of cell) (inside cell) bat bacteriorhodopsin (bR) LIGHT ADP + P ArgH L-arginosuccinate ArgG arginine ArcA ornithine carbamate ATP CO 2 ArcB ArcC citrulline carbamoyl-PO4 ADP + P ATP NH3 Why is it so complicated? • Why does the cell use this network of genes, enzymes, other proteins, and metabolites to make bR? • Why does the cell go through the trouble of regulating the network for bR? What could we do to test our network model? • How could we validate our network? • What other types of experiments could we do? • What other types of information could we use?