Abstract

Gene expression during lytic development of bacteriophage Mu is regulated by a
transcriptional cascade in three phases: early, middle and late. Transcription from the
middle promoter P_m requires the 129-amino acid transcriptional activator Mor, a product
of early transcription, and the Escherichia coli RNA polymerase. The P_m promoter has a
recognizable -10 hexamer but lacks a -35 hexamer. Mor binds as a dimer to an imperfect
dyad-symmetrical element containing two 6-bp inverted repeats and centered at -43.5 in
P_m. The goals of this study were:

1. To test the prediction from the crystal structure of Mor that residues Y70 and Q68
of the β-strand whose side chains extend away from the protein, make base
specific interactions in the DNA minor groove.
2. To identify the bases between -30 and -57 of the promoter P_m that are important
for its function, in terms of binding to His-Mor and transactivation with
interactions with RNAP subunits, thus optimizing the Mor-binding sequence of
Pm for crystal structure determination for a Mor-DNA duplex.
3. To identify the critical number of bases of P_m required for the best binding of Mor
and to test the stability of Mor binding to DNA probe containing P_m.

To test the prediction of base specific interactions of the side chains of Y70 and
Q68, mutagenesis of the 4-bp spacer region in the minor groove of the Mor-binding site
using degenerate oligonucleotides were done to introduce all possible substitutions. Plate
phenotyping on MacConkey agar plates was used to select P_m-lacZ clones that gave a
defective phenotype indicated by white color. All the white ones had other mutations
elsewhere in the plasmid and all the identified substitutions gave functional phenotypes
as indicated by red color of colonies. This experiment revealed that the specific bases in
the minor groove are not extremely important for interaction with the side chains of Y70
and Q68 as they can tolerate mutations. But, gel shift and β-galactosidase expression data
with a subset of these mutations indicated that the bases of the minor groove spacer
region do play a modest role in His-Mor binding and activation of P_m as visible from
their variations in the binding and tranactivation assay.

Specific mutations were introduced in the P_m sequence from positions -30 through
-57 upstream of the transcription start site to identify the critical bases for Mor-P_m
interactions. Since Mor binds as a dimer to P_m, the mutations would indicate whether
symmetry of the positions with respect to -43.5 or the specificity of the bases is what
determines the importance of the bases at the respective positions. It would also help
identify mutations that could increase Mor binding and positions that could contribute to
interaction with RNAP subunits. Plate phenotyping, in vitro binding assay and in vivo ?-
galactosidase assays were done for all the mutations. The 6-bp imperfect dyad-
symmetrical sequences flanking the minor groove spacer were found to be the most
critical for Mor binding. Within the dyad-symmetry element, bases at positions -38 to -40
and -47 to -49 are the most important as they do not tolerate any base changes. The
region flanking the Mor-binding sites on either side does not seem to be critical for Mor
binding, but the results indicate their function in transactivation, probably by influencing
interactions of Mor with the RNAP subunits or conformational changes in the
interactions at Mor-DNA-RNAP interfaces.

Two mutations, -46C alone and in combination with -50T, were specifically
interesting as they bound to wild-type His-Mor more effectively than wild-type promoter,
but displayed reduced in vivo activity. This observation led to the prediction that Mor
also functions in promoter clearance and that higher binding of Mor to the promoter
somehow negatively affects release of the core RNAP for transcription. This could mean
that Mor has dual functions at the middle promoter: recruitment of RNAP and release of
core RNAP during transcription initiation. Oligonucleotides with these specific mutations
can be used for crystal structure determination for a Mor-DNA duplex as they stabilize
the complex.
Different length oligonucleotides were used in gel shift assays with wild-type His-
Mor to identify the critical number of bases needed for efficient Mor binding. This
experiment revealed that at least 20-bp, centered at -43.5, is needed for detectable binding
to His-Mor.

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Revised 24 July 2008