Escherichia coli purine repressor: Regulation and structure-function analysis

Kang-Yell Choi, Purdue University

Abstract

The Escherichia coli purine repressor (PurR) is a transcriptional regulator for genes involved in purine nucleotide biosynthesis and related pathways. Chemical cross-linking studies show that PurR is a homodimer. Limited proteolysis identified two major structural domains of PurR; an N-terminal 52 amino acid domain for DNA binding and a 284 residue C-terminal domain for corepressor binding and dimerization. These two domains are connected by a short exposed hinge. The 284 amino acid C-terminal domain is homologous to periplasmic sugar binding proteins for arabinose, galactose and ribose. The corepressors, hypoxanthine and guanine, bind cooperatively to each subunit with affinities of 9.3 and 1.5 $\mu$M, respectively. Upon binding of corepressors, PurR undergoes a conformational change that increases its binding affinity for operator DNA. Amino acid residues required for corepressor binding were inferred by sequence comparison of PurR with periplasmic sugar binding proteins of known structure as well as other LacI family repressors, and subsequently replaced by site directed mutagenesis. The analysis of PurR mutants indicates that Asp 146, Trp 147, Arg 196 and Asp275 function in binding the corepressors. Conservation of ligand binding residues between PurR and sugar binding proteins suggests the overall similarity of the PurR corepressor binding domain with sugar binding proteins and leads to the proposal that effector sites must be similar among these proteins. The four mutations constructed perturbed the binding of both hypoxanthine and guanine and thus provides evidence for a single corepressor site per PurR subunit. A significant conformational change of the protease hypersensitive hinge of PurR was detected by binding of operator DNA. To understand the role of the hinge, 16 different mutations in four central hinge residues were produced and characterized. Mutations in the hinge region impaired repressor function in vivo. Several nonfunctional hinge mutants were defective in low affinity binding to operator DNA in the absence of corepressor as well as in high affinity corepressor-dependent binding to operator DNA, although binding of corepressor and general structural characteristics were similar to the wild type PurR. These results establish a role for the hinge region in operator binding and together with the studies of corepressor binding mutants lead to a proposal for two routes to form the holoPurR-operator complex: (i) interaction of apoPurR with operator followed by binding of corepressor to the apoPurR-operator complex and (ii) binding of holoPurR to operator. Homology of PurR and LacI may account for the effector-independent binding to operator DNA. (Abstract shortened by UMI.)

Degree

Ph.D.

Advisors

Zalkin, Purdue University.

Subject Area

Molecular biology|Microbiology|Genetics

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