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oai:www.ideals.illinois.edu:2142/19066 2023-07-10 col_2142_5131 col_2142_16338 com_2142_5130 com_2142_16337 com_2142_14793 com_2142_8903

Gardner, Jeffrey F. Hales, Laura Marie 2011-05-07T11:55:55Z 2011-05-07T11:55:55Z 10000-01-01 1995 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. Made available in DSpace on 2011-05-07T11:55:55Z (GMT). No. of bitstreams: 2 license.txt: 4922 bytes, checksum: 910b249b4beec47e7ab768910c8f966f (MD5) 9624356.pdf: 9394502 bytes, checksum: 1c6687198d8cb93a9700c7bd7a12e486 (MD5) Previous issue date: 1995 Item marked as restricted to the 'UIUC Users [automated]' Group (id=2) by Howard Ding (hding2@illinois.edu) on 2011-05-07T14:34:26Z Item is restricted indefinitely. Restriction data tranferred 2014-07-01T11:13:08-05:00 Original Data Group with Access UIUC Users [automated] Release Date: none Reason: ETDs are only available to UIUC Users without author permission ETDs are only available to UIUC Users without author permission U of I Only AAI9624356 (UMI)AAI9624356 http://hdl.handle.net/2142/19066 eng Copyright 1995 Hales, Laura Marie Biology, Molecular Biology, Microbiology A study of Escherichia coli integration host factor interaction with DNA text Microbiology Microbiology University of Illinois at Urbana-Champaign Dissertation Ph.D.