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Title:Genetic analysis of lambda integrase and Escherichia coli integration host factor: Interactions with their recognition sites
Author(s):Lee, Eunhee Cho
Doctoral Committee Chair(s):Gardner, Jeffrey F.
Department / Program:Microbiology
Degree Granting Institution:University of Illinois at Urbana-Champaign
Subject(s):Biology, Molecular
Biology, Genetics
Biology, Microbiology
Abstract:To study integrase (Int) binding to its arm-type sequences, bacteriophage P22-based challenge phages were constructed that carried either the contiguous P$\sp\prime$123 arm-type sites or the single P$\sp\prime$1 site within the P22 P$\sb{ant}$ promoter. Int was produced from a plasmid where its synthesis was regulated by the inducible P$\sb{tac}$ promoter. Int repressed P$\sb{ant}$ in phages containing the P$\sp\prime$123 sites more efficiently than phages carrying the single P$\sp\prime$1 site, suggesting that the protein binds cooperatively at the three adjacent P$\sp\prime$123 sites. The Int protein from a related lambdoid phage, HK022, also repressed transcription by binding to the same arm-type sites. Mutants with changes in the P$\sp\prime$123 sites or P$\sp\prime$1 sites that impair Int binding were isolated by selecting phages that expressed antirepressor in the presence of Int. DNA sequence analyses showed that most of the base pairs in the conserved recognition sequences are involved in the sequence specific recognition by Int protein.
The same strategy was utilized to isolate mutants containing changes in the H$\sp\prime$ sequence that decrease or eliminate IHF binding. Approximately one-half of the mutants contained substitutions that changed base pairs that are part of the IHF consensus sequence. Other mutants contained changes at base-pairs between the two subdeterminants of the H$\sp\prime$ site, at positions that are not specified in the consensus sequence, and in the dA-dT rich region that flanks the consensus region of the site. These results show that single base-pair changes at positions outside of the proposed consensus bases can weaken or drastically disrupt IHF binding to the mutated site.
Mutants encoding IHF proteins with altered DNA binding specificities were isolated by selecting for mutants which recognize one of the variant H$\sp\prime$ sites. Two IHF mutants recognized the H$\sp\prime$ variant containing a change at position 38 in the H$\sp\prime$ site. They carried single substitutions of either a proline to a leucine at residue 64 or a lysine to a serine at residue 65 in the $\alpha$ subunit. Three different IHF mutants replaced the glutamic acid at residue 44 in the $\beta$ subunit by a lysine, a valine, or a glycine. These three variants acquired the ability to bind to the mutant H$\sp\prime$ site containing a change at position 44 in the H$\sp\prime$ site. These results suggest that the domains of the $\alpha$ and $\beta$ subunits that contain the suppressor amino acids are involved in sequence specific recognition, either directly or indirectly.
Issue Date:1991
Rights Information:Copyright 1991 Lee, Eunhee Cho
Date Available in IDEALS:2011-05-07
Identifier in Online Catalog:AAI9136646
OCLC Identifier:(UMI)AAI9136646

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