Interaction of Bacteriophage Mu Middle Transcription Activator Protein Mor with Promoter DNA
type: CreativeWork
description: 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 wit…
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 wit…
author: Iyer, Kartik
keywords: Bacteriophage Mu, Mu middle promoter Pm, transcription activator protein Mor, promoter-activator interactions, Mor-DNA interactions, promoter mutations, Medical Immunology, Medical Sciences, Medicine and Health Sciences
date published: 2008-12-01T08:00:00Z
date modified: 2026-07-24T05:00:23Z
provider: University of Tennessee Health Science Center
publisher: Networked Digital Library of Theses and Dissertations
type: WebPage
source organization: University of Tennessee Health Science Center