Cellular metabolism includes a multitude of processes essential for the maintenance of life. Studies of metabolism in procaryotes and eucaryotes have clearly established that regulation of enzyme synthesis and the control of activity of preexisting enzymes are two basic mechanisms for adjusting the rates of metabolic reactions in the living cell. The long- term objective of this research is to identify, study and establish novel mechanisms in metabolic regulation at both the protein and the gene level. The inducible biodegradative threonine dehydratase (tdc) operon of Escherichia coli, involved in anaerobic energy metabolism, is an ideal prototype model system to investigate global and operon-specific controls of gene expression and molecular basis of enzyme regulation. A combination of biochemical and molecular genetic techniques will be used to analyze the transcriptional regulation of the tdc genes, characterize their gene products, and examine the molecular architecture of threonine dehydratase and its interactions with allosteric modifiers. Specifically, experiments are proposed to purify the tdcR gene product (an activator protein) and localize its binding site on the tdc promoter for trans-activation of the tdc operon; identify the DNA sequence recognized by the RNA polymerase-sigma54 to deduce its function in mRNA transcription; clone the gene(s) involved in inducer synthesis by in vivo complementation of the inducer-minus phenotype; characterize threonine transport by the tdcA gene product; and isolate mutant forms of threonine dehydratase by site-directed mutagenesis with altered active and regulatory sites to examine the structural requirements for enzyme catalysis and its regulation by cellular metabolites. It is hoped that this integrated knowledge of the genes and proteins in anaerobic threonine degradation will provide new insight into the basic metabolic processes in the living cell and how loss of normal regulatory controls may trigger metabolite imbalance resulting from biochemical and genetic disorders.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM021436-16
Application #
3270477
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1975-03-01
Project End
1995-02-28
Budget Start
1990-03-01
Budget End
1991-02-28
Support Year
16
Fiscal Year
1990
Total Cost
Indirect Cost
Name
University of Michigan Ann Arbor
Department
Type
Schools of Medicine
DUNS #
791277940
City
Ann Arbor
State
MI
Country
United States
Zip Code
48109
Chattopadhyay, S; Wu, Y; Datta, P (1997) Involvement of Fnr and ArcA in anaerobic expression of the tdc operon of Escherichia coli. J Bacteriol 179:4868-73
Wu, Y; Datta, P (1995) Influence of DNA topology on expression of the tdc operon in Escherichia coli K-12. Mol Gen Genet 247:764-7
Hagewood, B T; Ganduri, Y L; Datta, P (1994) Functional analysis of the tdcABC promoter of Escherichia coli: roles of TdcA and TdcR. J Bacteriol 176:6214-20
Ganduri, Y L; Sadda, S R; Datta, M W et al. (1993) TdcA, a transcriptional activator of the tdcABC operon of Escherichia coli, is a member of the LysR family of proteins. Mol Gen Genet 240:395-402
Wu, Y; Patil, R V; Datta, P (1992) Catabolite gene activator protein and integration host factor act in concert to regulate tdc operon expression in Escherichia coli. J Bacteriol 174:6918-27
Wu, Y F; Datta, P (1992) Integration host factor is required for positive regulation of the tdc operon of Escherichia coli. J Bacteriol 174:233-40
Schweizer, H P; Datta, P (1991) Physical linkage and transcriptional orientation of the tdc operon on the Escherichia coli chromosome. Mol Gen Genet 228:125-8
Sumantran, V N; Schweizer, H P; Datta, P (1990) A novel membrane-associated threonine permease encoded by the tdcC gene of Escherichia coli. J Bacteriol 172:4288-94
Schweizer, H P; Datta, P (1989) Identification and DNA sequence of tdcR, a positive regulatory gene of the tdc operon of Escherichia coli. Mol Gen Genet 218:516-22
Patil, R V; Datta, P (1989) Amino acid sequence of the regulatory-site glyoxylate peptide of biodegradative threonine dehydratase of Escherichia coli. J Bacteriol 171:3379-84

Showing the most recent 10 out of 17 publications