Date of Award

12-2007

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Microbiology and Immunology

Research Advisor

Martha M. Howe, Ph.D.

Committee

Hee Won Park, Ph.D. Marko Radic, Ph.D. Rajendra Raghow, Ph.D. James Patrick Ryan, Ph.D

Abstract

The lytic cycle of phage Mu is regulated by a transcriptional cascade consisting of early, middle and late transcription. The Mor protein is an activator of the middle promoter Pm and is encoded by the last gene of the early transcript. The C protein is an activator of the four late promoters Plys, PI, PP, and Pmom and is expressed from the middle transcript. Both Mor and C proteins bind an imperfect dyad-symmetry element just upstream and overlapping the –35 region of Pm and Plys respectively. The main aims of this study was, (1) To understand the binding specificity of C and determine a possible consensus sequence for C binding, and (2) To crystallize the C : DNA complex as a first step towards structure determination. In previous work, single base substitution mutations in Plys identified bases and positions important for C binding and activation. To get a consensus sequence for C binding, we tested additional candidate mutations within and flanking the C binding sequence. Wild-type C protein was used in gel mobility shift assays with annealed oligonucleotides containing mutations, insertions and deletions. The assay showed that, (1) mutation in positions –53, –52 and –32 did not affect C binding, (2) mutations flanking the IR spacer (–40, –41, –46, –47) influence C binding, and (3) insertion or deletion of a single base pair in the IR spacer abolished C binding. Mor and C proteins are the founding members of a new class of transcription factors. The Mor structure revealed that it has a classical DNA-binding HTH motif and a dimerization domain. Based on the structure it has been proposed that Mor has to undergo conformational changes to bind DNA. Modelling of C based on the Mor structure revealed that C might also have a dimerization domain and a HTH DNA binding motif. To see if any conformational changes occur in C when it binds DNA, co- crystallization of a C : DNA complex was undertaken. Preliminary structural analysis of the complex revealed that under the crystallization conditions used C protein is bound to its symmetrical binding site using two HTH motifs from two C dimers without inducing any conformational change in itself or the DNA.

DOI

10.21007/etd.cghs.2007.0283

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