Characterization of the regulatory components required for genetic regulation of purine nucleotide biosynthesis in Escherichia coli

Ronda Joan Rolfes, Purdue University

Abstract

In Escherichia coli, the genes encoding the enzymes for the de novo synthesis of purine nucleotides are scattered throughout the chromosome in small clusters and single cistrons. Addition of preformed purines represses expression of these pur genes. The cis-acting sequences required for the expression and control of the gene purF were probed by site-directed mutagenesis. By this analysis, the operator and $-$35 promoter elements were identified and the assignment of the $-$10 promoter sequence was confirmed. Spontaneously occurring mutants in purR were isolated based on increased lactose utilization in a purF-lacZ fusion under purine repression conditions. The purR$\sp+$ gene was isolated by complementation of purR utilizing a mini-Mu library. The cloned gene encodes a protein, Pur repressor, of 341 amino acids. Pur repressor is homologous to the Lac, Gal, Cyt, Mal, Raf and Rbs repressors. Pur repressor utilizes a helix-turn-helix binding motif. Pur repressor bind to the purF operator but not to DNA containing an operator-constitutive mutation. Pur repressor protects a 16-base pair palindromic sequence, ACGCAAAC $\cdot$ GTTTTCTT, from DNaseI digestion. Two sequences similar to the pur operator were discovered in purR: both are downstream of the transcription start, one is within the coding region. The role of these operator-like sequences was investigated in vitro by DNA binding assays, and in vivo by measurements of $\beta$-galactosidase activities and RNA levels of purR-lacZ fusions. Pur repressor binds noncooperatively to both operators with different affinities. Analysis of wild-type and mutant operators demonstrated that both are required for in vivo autogenous regulation. The purR gene was subcloned behind the T7 promoter for overexpression. Pur repressor was purified to homogeneity by fractionation on DEAE-Sepharose followed by Heparin-Agarose. Edman degradation revealed that the amino-terminal methionine is cleaved. In vitro DNA binding assays using purified Pur repressor and purF operator DNA were used to identify corepressors. Half-maximal binding of repressor to DNA occurred at a guanine concentration of 1.7 $\mu$M and a hypoxanthine concentration of 7.1 $\mu$M. The K$\sb{\rm d}$(app) of repressor for purF operator is 3.4 nM.

Degree

Ph.D.

Advisors

Zalkin, Purdue University.

Subject Area

Biochemistry|Molecular biology

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