Integration Host Factor and Its Interaction With the H-Prime Site of Bacteriophage Lambda

Integration Host Factor (IHF) is a sequence-specific DNA-bending protein of Escherichia coli that recognizes the DNA through contacts made to the backbone and the minor groove. IHF binds specifically to the H$\sp\prime$ site, $5\sp\prime$-TAAAAAAGCATTGCTTATCAATTTGTTGCAACGA-$3\sp\prime$, with a $\rm K\sb{d}(app)$ of 1 nM. Substitution of an A for the G within the TTG element, which is allowable in the consensus, increases the $\rm K\sb{d}(app)$ to 10 nM and indicates that context is important in the recognition of this conserved position. Any other single base substitution within the TTG element increases the Kd(app) of IHF for the H$\sp\prime$ site to ${>}2750$ nM and shows that the interaction of IHF with the TTG sequence is very specific. By combining the genetic data of these studies with the structural data of the IHF-H$\sp\prime$ site cocrystal the specificity can be explained as a combination of direct and indirect readout of sequence information. Mutants that remove the glutamic acid 44 of the β-subunit restore wild-type levels of IHF binding to an H$\sp\prime$ site containing a TAG element. These mutants still bind the wild-type TTG-containing H$\sp\prime$ site efficiently. However, these mutants do not allow IHF to recognize other mutant variants of the H$\sp\prime$ site. Substitution with a variety of amino acid sidechains demonstrates that both oxygens of the glutamic acid sidechain are required for full discrimination against the TAG variant. In the cocrystal, the oxygens of the glutamic acid make bonds with two flanking arginines but do not contact the DNA. These results also demonstrate that IHF can distinguish between a T:A and A:T basepair through interactions in the minor groove.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Microbiology

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2015

Author dc:creator

• Read, Erik Karl

Contributors dc:contributor

• Gardner, Jeffrey F.

• Biology, Molecular

Language dc:language

eng

Identifier

(MiAaPQ)AAI9812746

OAI identifier oai:identifier

oai:www.ideals.illinois.edu:2142/86725