Date of Award

12-2008

Document Type

Thesis

Degree Name

Master of Science (MS)

Program

Biomedical Sciences

Track

Microbial Pathogenesis, Immunology, and Inflammation

Research Advisor

Martha M Howe, Ph.D.

Committee

James E. Bina, Ph.D. Susan E. Senogles, Ph.D.

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 Pm requires the 129-amino acid transcriptional activator Mor, a product of early transcription, and the Escherichia coli RNA polymerase. The Pm 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 Pm. 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 Pm 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 Pm required for the best binding of Mor

and to test the stability of Mor binding to DNA probe containing Pm.

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 Pm-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 Pm as visible from their variations in the binding and tranactivation assay.

Specific mutations were introduced in the Pm sequence from positions -30 through

-57 upstream of the transcription start site to identify the critical bases for Mor-Pm

interactions. Since Mor binds as a dimer to Pm, 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 β- galactosidase assays were done for all the mutations. The 6-bp imperfect dyadsymmetrical 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.

DOI

10.21007/etd.cghs.2008.0147

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