Date of Award
Master of Science (MS)
Biochemistry and Molecular Biology
College of Graduate Studies
The bacteriophage T4 regA protein translationally represses over 30 T4 early genes, including the regA gene, by binding to the translation initiation region of the T4 mRNAs (Karam et al., 1981). Although the recognition sequence has been at least partially characterized in a number regA sensitive mRNAs, there is no consensus sequence or structural feature that is recognized by regA in all transcripts (Winter et al., 1987; Webster et al., 1989; Unnithan et al., 1990). Recent studies have identified an RNA binding site on regA protein, but there remains a large gap in our understanding of how regA protein functions to temporally repress multiple mRNAs (Gordon et aI., 1999). A genetic screen was developed to further identify regA protein:RNA interaction sites. This approach uses a two-plasmid system in which lacZ expression is placed under the translational control of T4 regA via the gene 44 recognition element (gene 44 RE) (AAUGAGGAAAUU) on plasmid pLacZ-44RE. For regA expression the regA gene was cloned into a pACYC derivative thereby placing expression of regA under the control of a strong IPTG inducible promoter. The two plasmids were then transformed into the E. coli strain WM1/F'. This approach allowed the rapid selection of regA mutants with altered RNA binding affinity using a blue/white selection process. After selection, the regA mutants were further characterized by sequencing of the mutant genes, as well as by gel mobility shift assays with the mutant proteins and an RNA oligo of the gene 44 RE. Beta-galactosidase assays were also performed to quantify repression levels obtained by IPTO induction of regA in cells transformed with the mutant regAs. In subsequent experiments, the gene 44 RE was mutated at two of the twelve bases in order to vary regA affinity for the binding site and thereby vary regA repression of the lacZ mRNA translation. In another experiment the Shine-Dalgamo sequence upstream of the lacZ gene was weakened to reduce b-galactosidase expression. Blue/white assays were performed to determine if these base mutations produced variations in color due to variable expression of beta-galactosidase. After comparing lacZ expression from plasmids with the various gene 44 RE mutants, the regA gene was randomly mutagenized and the blue/white screen was used to look for "gain of function" mutants. Potential mutants were expected to be altered in their RNA binding specificity. The mutant colonies were sequenced to identify specific mutations, in an effort to identify residues that are important in the recognition and binding of RNA targets. The effects of individual mutations were further assayed by the use of gel mobility shift and β-galactosidase assays. In other experiments color screening assays were performed to compare cells expressing T4 regA with cells expressing the regA homologue found in bacteriophage RB69. Previous studies had indicated that RB69 regA had a greater affinity for RNA than did T4 regA, in vitro. The in vivo color assays were performed to determine if the higher affinity in vitro translated into greater repression of lacZ in vivo. Results from these screens indicate that the hierarchy of repression of lacZ expression is from plasmids with different gene 44 REs is different for T4 regA and RB69 regA proteins. The work described in Chapter II of this thesis is concerned with the aryl hydrocarbon receptor protein (AHR). Recent studies have shown that the AHR protein is rapidly degraded both in vivo and in vitro after exposure to agonist (Pollenz, R.S., 1996; Giannone et al., 1998; Giannone et al., 1995; Pollenz et al., 1998; Roman et al., 1998; Sommer et al., 1998). Additionally, it has been shown that the AHR contains putative Nuclear Export Signal (NES) and Nuclear Localization Signal (NLS) sequences (Davarinos and Pollenz, 1999; Ikuta et al., 1999; Pollenz and Barbour, 2000). The purpose of the studies detailed in this thesis was to generate plasmids which express AHR mutant proteins that contain mutant NES or NLS sequences but are unimpaired in all other aspects of AHR functionality. These studies have shown that it is possible to generate AHR proteins with mutations in the NES that dimerizes with ARNT, binds DNA and supports ligand-mediated gene induction. These studies have also shown that AHR proteins with mutant NLS sequences are also capable of dimerizing with ARNT and binding to DNA. Additionally, these studies have indicated that the NLS mutants were degraded more slowly after agonist exposure than wild type AHR suggesting another level of regulation of the AHR pathway.
Barbour, Estel Rick, "In Vitro Mutagenesis for Structure/Function Studies of the Bacteriophage T4 Rega Protein and the Murine AHR Protein" (2000). MUSC Theses and Dissertations. 87.
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