In biological systems, most processes are under the control of a variety of regulatory mechanisms. For example, the glyoxylate bypass of Escherichia coli is controlled by regulation of the expression of the glyoxylate bypass operon, by the phosphorylation of isocitrate dehydrogenase (IDH) and by allosteric mechanisms. Our long term goal is to characterize the functioning and coordination of these regulatory mechanisms. This proposal focuses on the regulation of expression of the glyoxylate bypass operon and the mechanism(s) responsible for differential expression of its products. The glyoxylate bypass operon encodes the regulatory and metabolic enzymes of the glyoxylate bypass. This bypass is catalyzed by isocitrate lyase (aceA) and malate synthase (aceB) and is regulated by IDH kinase/phosphatase (aceK). The operon is fully induced only during growth on acetate and is repressed if any preferred carbon source is simultaneously present. A goal of this project is to determine the mechanism(s) by which the expression of this operon is controlled. Under this proposal, we will focus primarily on the products of two genes which may encode repressor proteins, iclR and fadR. Although aceA is immediately upstream of aceK, the cellular level of isocitrate lyase is 100 to 1,000 fold greater than that of IDH kinase/phosphatase. One of the goals of this proposal is to identify the mechanism(s) which is responsible for this downshift in expression. Our working model proposes that the downshift in expression results from inefficient translational initiation of aceK, possibly as a result of mRNA secondary structure. Premature transcriptional termination and/or instability of the aceK region of the mRNA also appear to contribute to this downshift in expression and may be secondary consequences of inefficient translational initiation. A variety of approaches will be used to test this and other models which are described in the proposal.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK040486-03
Application #
3240793
Study Section
Physiological Chemistry Study Section (PC)
Project Start
1988-09-01
Project End
1991-08-31
Budget Start
1990-09-01
Budget End
1991-08-31
Support Year
3
Fiscal Year
1990
Total Cost
Indirect Cost
Name
University of Minnesota Twin Cities
Department
Type
Schools of Medicine
DUNS #
168559177
City
Minneapolis
State
MN
Country
United States
Zip Code
55455
Miller, S P; Chen, R; Karschnia, E J et al. (2000) Locations of the regulatory sites for isocitrate dehydrogenase kinase/phosphatase. J Biol Chem 275:833-9
Finer-Moore, J; Tsutakawa, S E; Cherbavaz, D R et al. (1997) Access to phosphorylation in isocitrate dehydrogenase may occur by domain shifting. Biochemistry 36:13890-6
Pan, B; Unnikrishnan, I; LaPorte, D C (1996) The binding site of the IclR repressor protein overlaps the promoter of aceBAK. J Bacteriol 178:3982-4
Resnik, E; Pan, B; Ramani, N et al. (1996) Integration host factor amplifies the induction of the aceBAK operon of Escherichia coli by relieving IclR repression. J Bacteriol 178:2715-7
Gui, L; Sunnarborg, A; LaPorte, D C (1996) Regulated expression of a repressor protein: FadR activates iclR. J Bacteriol 178:4704-9
Gui, L; Sunnarborg, A; Pan, B et al. (1996) Autoregulation of iclR, the gene encoding the repressor of the glyoxylate bypass operon. J Bacteriol 178:321-4
Miller, S P; Karschnia, E J; Ikeda, T P et al. (1996) Isocitrate dehydrogenase kinase/phosphatase. Kinetic characteristics of the wild-type and two mutant proteins. J Biol Chem 271:19124-8
LaPorte, D C (1993) The isocitrate dehydrogenase phosphorylation cycle: regulation and enzymology. J Cell Biochem 51:14-8
Chung, T; Resnik, E; Stueland, C et al. (1993) Relative expression of the products of glyoxylate bypass operon: contributions of transcription and translation. J Bacteriol 175:4572-5
Firestone, J A; Browning, M D (1992) Synapsin II phosphorylation and catecholamine release in bovine adrenal chromaffin cells: additive effects of histamine and nicotine. J Neurochem 58:441-7

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