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.

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Revised 06 August 2007