Construction and characterization oftrpR-based chimaeric genes and their application to the analysis of regulatory mechanisms

Jill Helen Zeilstra-Ryalls, Purdue University

Abstract

A plasmid has been constructed to express and secrete Trp repressor in yeast. It was demonstrated that Trp repressor is secreted into the culture media to a final concentration of approximately 1 mg/l. With modifications, this system should provide experimental material for investigating in detail which E. coli proteins interact with Trp repressor. An unusual feature of the trpR gene, a G/C-rich palindrome located in the region upstream of the trpR coding sequence, has long been the subject of speculation regarding its potential contribution to the level of expression of trpR. Using the products of in vivo and in vitro DNA manipulations, it has been determined that this palindrome does not significantly affect the level of expression; rather, it appears that the Shine-Dalgarno sequence plays a significant role in determining the intracellular levels of Trp repressor. Using a subset of the "biological tools" developed for the investigation of regulatory features of the trpR gene, an in vivo structural analysis of donor peptide in the $\alpha$-complementation reaction of $\beta$-galactosidase was executed. The $\alpha$-donors that were analyzed consisted of truncated derivatives of chimaeric Trp repressor$\sp\prime$-$\sp\prime$LacZ polypeptides. It was found that chimaeric $\alpha$-peptides containing greater amounts of $\sp\prime$lacZ$\alpha$ coding sequence (residues 9-146) have more latitude with respect to the nature of the N-terminus than homologous $\alpha$-peptide chimaeras with lesser amounts of $\sp\prime lacZ\alpha$ coding sequence (residues 9-51). It has also been possible to demonstrate, in vivo, an interaction between the chimaeric $\alpha$-donor peptides and the $\alpha$-acceptor protein that takes place independently of the formation of catalytically active $\beta$-galactosidase. A genetic system designed to obtain trpR mutants having mutations in residues critical to the formation of Trp repressor dimers was developed. This system involved, inter alia, the construction of a trpR$\sp\prime$-$\sp\prime$tetA fusion and a trpR$\sp\prime$-$\sp\prime$nptII fusions. The Trp repressor$\sp\prime$-$\sp\prime$TetA chimaeric protein has operator binding activity. The Trp repressor$\sp\prime$-$\sp\prime$NptII protein is less well-characterized owing to the fact that the protein undergoes post-translational processing. Investigation of the properties of these chimaeric proteins has helped define certain novel aspects of the structure-function relationships applicable to Trp repressor, for example the degree of sequence flexibility tolerated within the DNA "specificity helix" (Schevitz et al., 1985), that many now be investigated by genetic and biochemical approaches.

Degree

Ph.D.

Advisors

Somerville, Purdue University.

Subject Area

Molecular biology

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