Escherichia coli tryptophan repressor binds multiple sites within the aroH and trp operators.

Abstract

The Trp repressor of Escherichia coli coordinately regulates the expression of the trp, aroH, and trpR operons in response to the intracellular levels of L-tryptophan (Cohen and Jacob 1959;Brown 1968;Rose et al. 1973;Gunsalus and Yanofsky 1980). The trpR operon, located at 99.5 min, codes for the Trp aporepressor and is autoregulated (Morse and Yanofsky 1969;Gunsalus and Yanofsky 1980;Kelley and Yanofsky 1982;Bachman 1983;Gunsalus et al. 1986). The aroH operon, located at 37 min, encodes one of the three isozymes of 3-deoxy-D-arabino-heptulosonic acid-7-phosphate synthetase (DAHP). This enzyme catalyzes the first step in the common pathway of aromatic amino acid biosynthesis (Brown 1968;Pittard et al. 1969;Zurawski et al. 1981). The third operon, trp, is located at 27 min on the E. coli genetic map and encodes the enzymes that carry out the final steps of L-tryptophan biosynthesis from the cellular intermediate, chorismic acid (Yanofsky 1971).The native form of the Trp aporepressor is a dimer composed of identical subunits of 108 amino acids (Gunsalus and Yanofsky 1980;Joachimiak et al. 1983;Arvidson et al. 1986). Binding of the corepressor, L-tryptophan increases the affinity of Trp repressor for operator DNA, leading to the formation of the ternary repressor DNA complex (Rose et al. 1973). The binding of Trp repressor to its operators inhibits the initiation of transcription of the trpR, aroH, and trp operon messages by preventing RNA polymerase from binding to the promoters (Oppenheim et al. 1980).The physical location of the trp operator was previously defined by deletion studies (Bennett et al. 1976) and by the isolation of constitutive mutations within the trp operator {Bennett and Yanofsky 1978). These operator constitutive mutations map at positions -16, -15, -7, and -6 in the trp promoter sequence. The DNA sequences of the trpR and aroH operons reveal sites that exhibit significant sequence homology to the trp operator (Gunsalus and Yanofsky 1980; Singelton et al. 1980; Zurawski et al. 19811. In Figure 1, the sequences of the three operators are aligned to show max- • .• . .• .• ::::~ -20 .....

DNA specificity determinants of Escherichia coli tryptophan repressor binding.

“…Consistent with these results, Bennett and Yanofsky (1978)have isolated constitutive mutations at positions + 4, + 5, and -6 within the trp operator half-sites that represent four of these eight most critical changes. The G : C base pairs in the natural operators implicated in binding by Kumamoto et al (1987) include the same G:C base pairs identified as important by our genetic method.…”

“…Our results, taken together with the crystal structure of Trp repressor (Schevitz et al 1985) and data from chemical protection experiments that define the interaction of Trp repressor with naturally occurring operators (Kumamoto et al 1987), enable us to construct a model for the specificity determinants of the Trp repressor/operator interaction.…”

“…The most critical base pairs for repressor binding in each operator half-site are indicated in boldface. Presumably, three Trp repressor dimers contact successive pairs of major grooves of the trp operator, and two dimers bind the aroH operator, adjacent to faces of the double helix that are rotated with respect to one another by about 270 ° (Kumamoto et al 1987).…”

“…The trp operator has three tandem sites, whereas the aroH operator has two sites. The trpR operator is the simplest of the three and is presumably bound by a single repressor dimer (Kumamoto et al 1987). An axis of twofold symmetry divides each of the six natural binding sites into two half-sites.…”

Advancing Our Knowledge in Biochemistry, Genetics, and Microbiology Through Studies on Tryptophan Metabolism

“…Lisa Klig and Barry Hurlburt of my group, with Tom Graddis and Dale Oxender at the University of Michigan, and independently Janette Carey of Princeton, identified many additional features of the trp repressor (72,73). After leaving my lab, Robert Gunsalus contributed the interesting finding that there appear to be multiple repressor binding sites in the trp operon operator region (74). Catherine Lawson and Janette Carey provided conclusive structural evidence in support of this proposition (75).…”