From structure to genetics and back : Using the LAC repressor to study protein-DNA interactions / Leslie Milk.

Format:

Book

Manuscript

Thesis/Dissertation

Author/Creator:

Milk, Leslie.

Contributor:

Lewis, Mitchell, advisor.

University of Pennsylvania.

Language:

English

Subjects (All):

Penn dissertations--Biochemistry and molecular biophysics.

Biochemistry and molecular biophysics--Penn dissertations.

Biochemistry and Molecular Biophysics.

Academic Dissertations as Topic.

Medical Subjects:

Biochemistry and Molecular Biophysics.

Academic Dissertations as Topic.

Local Subjects:

Penn dissertations--Biochemistry and molecular biophysics.

Biochemistry and molecular biophysics--Penn dissertations.

Physical Description:

viii, 186 pages : illustrations (some color) ; 29 cm

Production:

2010.

Summary:

In bacteria, the Lac repressor regulates expression of the genes required for lactose metabolism by binding a unique nucleotide sequence (operator) in response to lactose availability. The Lac operon has been adapted for use in various laboratory techniques and has been successfully used to exogenously regulate expression of target genes in mammals. It has extraordinary potential for use in therapeutics and as a tool for studying protein-DNA interactions.

Protein-DNA interactions are an integral component of essential cellular processes. Understanding the principles that dominate these interactions is necessary to manipulate these processes for laboratory or medicinal purposes. Despite significant contributions to the field of protein-DNA interactions, current understanding of protein- DNA interactions is not sufficient to predict functional amino acid-nucleotide combinations.

In this thesis, genetic screens were performed to identify the limits of Lac repressor-operator interactions. Amino acids that are involved in specific interactions with nucleotide bases were randomized and screened against operator variants. Results suggest that altering some (but not all) of these amino acids permit the Lac repressor to bind alternative operator sequences. The screens produced a functional code for Lac repressor-operator interactions and the largest set of protein-DNA binding partners, for a given scaffold, to date.

Structural studies were performed to visualize the interactions that occur when the Lac repressor is altered. The X-ray structure of the wild-type Lac repressor headpiece bound to the symmetric operator was solved, in addition to 5 mutant repressors bound to the same operator. The structures show that the Lac repressor is capable of accommodating modest mutations by preserving the overall structure of the complex but altering the amino acid-nucleotide contacts.

Taken together, the results of this thesis suggest that a complete understanding of protein-DNA interactions requires examination of the physical and chemical environment for every proposed interaction. Nuances of protein-DNA recognition cannot be simplified by rules that apply to protein families or even a single protein- DNA scaffold. The combination of the database and the crystallographic scheme will provide researchers with a tool to study protein-DNA interactions on an unprecedented level in the future.

Notes:

Adviser: Mitchell Lewis.

Thesis (Ph.D. in Biochemistry and Molecular Biophysics) -- University of Pennsylvania, 2010.

Includes bibliographical references.