Abstract

Genetic and biochemical experiments are described which will analyze the molecular interactions that control expression of the L-arabinose operon in Escherichia coli. The araBAD operon and araC regulatory gene promoters will be saturated with mutants using in vitro mutagenesis. The methods used will include (1) synthetic oligonucleotide-directed mutagenesis, (2) heavy mutagenesis with hydroxylamine, and (3) site-directed chemical modification. Mutations created in vitro will be transferred to the chromosome for analysis of their effect on expression of the ara genes. The possible functional significance of a putative polypeptide coded for by araC leader mRNA will be assessed. A comparison will be made at the DNA sequence level of ara promoters and the araC gene from other Enterobacteriaceae. The interaction of small molecules with purified araC regulatory protein will be studied by spectrofluorimetric methods. Finally, an attempt will be made to determine the 3-dimensional structure of the araC regulatory protein with and without L-arabinose and araC protein complexed with araI and araO DNA.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM030896-04
Application #
3278767
Study Section
(MG)
Project Start
1982-08-01
Project End
1987-07-31
Budget Start
1985-08-01
Budget End
1986-07-31
Support Year
4
Fiscal Year
1985
Total Cost
Indirect Cost

  Institution

Name
University of California Los Angeles
Department
Type
Schools of Arts and Sciences
DUNS #
119132785
City
Los Angeles
State
CA
Country
United States
Zip Code
90095

  Publications

Clarke, P; Lee, J H; Burke, K et al. (1992) Mutations in the araC gene of Salmonella typhimurium LT2 which affect both activator and auto-regulatory functions of the AraC protein. Gene 117:31-7
Nishitani, J; Wilcox, G (1991) Cloning and characterization of the L-rhamnose regulon in Salmonella typhimurium LT2. Gene 105:37-42
Stoltzfus, L; Wilcox, G (1989) Effect of mutations in the cyclic AMP receptor protein-binding site on araBAD and araC expression. J Bacteriol 171:1178-84
Burke, K A; Wilcox, G (1987) The araC gene of Citrobacter freundii. Gene 61:243-52
Lee, J H; Burke, K; Wilcox, G (1986) Mutations resulting in promoter-like sequences which enhance the expression of araC in Salmonella typhimurium. Gene 46:113-21
Cass, L G; Wilcox, G (1986) Mutations in the araC regulatory gene of Escherichia coli B/r that affect repressor and activator functions of AraC protein. J Bacteriol 166:892-900
Lei, S P; Lin, H C; Heffernan, L et al. (1985) araB Gene and nucleotide sequence of the araC gene of Erwinia carotovora. J Bacteriol 164:717-22
Lin, H C; Lei, S P; Wilcox, G (1985) The araBAD operon of Salmonella typhimurium LT2. I. Nucleotide sequence of araB and primary structure of its product, ribulokinase. Gene 34:111-22
Lin, H C; Lei, S P; Wilcox, G (1985) An improved DNA sequencing strategy. Anal Biochem 147:114-9
Lin, H C; Lei, S P; Wilcox, G (1985) The araBAD operon of Salmonella typhimurium LT2. II. Nucleotide sequence of araA and primary structure of its product, L-arabinose isomerase. Gene 34:123-8

Showing the most recent 10 out of 11 publications