Characterization of a set of integration host factor mutants deficient for DNA binding.
Integration host factor, IHF, is a sequence-specific DNA-binding and DNA-bending protein composed of two related but non-identical subunits. We report the isolation and characterization of hydroxylamine-induced loss-of-function mutations in the genes encoding the IHF subunits. To screen for mutants that preserve proper folding of IHF, clarified extracts were prepared from each mutant and were assayed for production of each subunit by immunoblotting and for formation of heterodimers by chemical cross-linking and subsequent immunoblotting. Extracts from mutants that met these criteria were found to bind a specific IHF site weakly if at all. These alleles therefore identify candidates for residues that may affect the DNA-binding surfaces of IHF. When projected onto the known tertiary structure of the closely related HU protein, these residues are found at the surface; however, with the exception of a single residue, different regions of the protein are implicated in each subunit. This suggests that, despite their homology, each subunit of IHF directs DNA recognition and binding in a distinct manner. To confirm the significance of the differential location of these mutations, we introduced in each subunit alterations that had been isolated as loss-of-function mutations at the corresponding position in the other subunit. In general, the engineered mutants have phenotypes that are strikingly different from those of their hydroxylamine-induced counterparts. In particular, most of the site-directed mutant IHF proteins form or maintain IHF:DNA complexes more readily than mutants that have the same change in the other subunit and were isolated as loss-of-function mutants. We discuss the positions of the mutant amino acid residues as they relate to a proposed molecular model of an IHF:DNA complex.
Granston
A E
AE
Laboratory of Molecular Biology, National Institute of Mental Health, Bethesda, MD 20892.
Nash
H A
HA
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D017931:DNA Primers / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005816:Genetic Complementation Test; D034982:Integration Host Factors; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011487:Protein Conformation; D013329:Structure-Activity Relationship
D001426:Bacterial Proteins; D017931:DNA Primers; D004268:DNA-Binding Proteins; D034982:Integration Host Factors; D046911:Macromolecular Substances