University of Illinois Urbana-Champaign

A study of Escherichia coli integration host factor interaction with DNA

Hales, Laura Marie

This item is only available for download by members of the University of Illinois community. Students, faculty, and staff at the U of I may log in with your NetID and password to view the item. If you are trying to access an Illinois-restricted dissertation or thesis, you can request a copy through your library's Inter-Library Loan office or purchase a copy directly from ProQuest.

Permalink

https://hdl.handle.net/2142/19066

Description

Title
A study of Escherichia coli integration host factor interaction with DNA
Author(s)
Hales, Laura Marie
Issue Date
1995
Doctoral Committee Chair(s)
Gardner, Jeffrey F.
Department of Study
Microbiology
Discipline
Microbiology
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
  • Biology, Molecular
  • Biology, Microbiology
Language
eng
Abstract
  • The sequence elements required for IHF binding to the H$\sp\prime$ and H1 sites in attP of $\lambda$ were examined. The H$\sp\prime$ site naturally contains a dA+dT element 5$\sp\prime$ to the core consensus element WATCAANNNNTTR, while the H1 site does not. It was found that both elements are required for IHF to bind to the H$\sp\prime$ site. In contrast, the core consensus determinant alone is sufficient for IHF binding to the H1 site. Placement of a dA+dT element upstream of the H1 core consensus element significantly increased the affinity, suggesting that the presence of a dA+dT element enhances IHF binding.
  • Mutants of IHF were examined for their ability to bind to various IHF binding sites in vitro and in vivo, and to promote recombination of $\lambda$ in vitro. The relative affinity of mutant IHF proteins was increased by the presence of the dA+dT region, confirming the enhancer-like properties of the dA+dT element. It was also found that these mutant proteins not only retain their DNA-bending ability but make any protein-protein contacts necessary to form a recombination-proficient intasome.
  • Gel mobility-shift assays using circularly permuted DNA fragments containing IHF sites showed that IHF forms a more compact protein-DNA complex with a site containing a dA+dT element when compared to a site lacking this sequence element. Additionally, artificial placement of a dA+dT element upstream of the H1 site in the genome of $\lambda$ weakened recombination in vitro. Therefore, the precise nucleoprotein structure required for $\lambda$ recombination is disrupted in this modified substrate. These results support the hypothesis that the DNA at an IHF site is bent in a different manner when the dA+dT element is present. It was demonstrated that IHF can form a specific protein-DNA crosslink with binding sites containing or lacking this dA+dT element. These results confirm the importance of flanking DNA and a dA+dT element in the binding and bending of a site by IHF.
Type of Resource
text
Permalink
http://hdl.handle.net/2142/19066
Copyright and License Information
Copyright 1995 Hales, Laura Marie

Owning Collections

Graduate Dissertations and Theses at Illinois PRIMARY
Graduate Theses and Dissertations at Illinois
Dissertations and Theses - Microbiology