HU-1 mutants of Escherichia coli deficient in DNA binding.
We constructed four mutants of the Escherichia coli hupB gene, encoding HU-1 protein, by synthetic oligodeoxyribonucleotide-directed, site-specific mutagenesis on M13mp18 vectors. The HupBR45 protein contained alterations of Arg58----Gly and Arg61----Gly, and the HupBF3, HupBK2 and HupBA1 proteins contained Phe47----Thr, Lys37----Gln and Ala30----Asp alterations, respectively. HupBF3 and HupBR45 were unable to maintain normal cell growth in a hupA-hupB-himA triple mutant at 42 degrees C, mini-F or RSF1010 proliferation, or Mu phage development in a hupA-hupB double mutant, whereas HupBA1 and HupBK2 supported these cellular activities. DNA-affinity column chromatography showed that the HupBF3 and HupBR45 had reduced affinities to DNA. These observations indicate that two highly conserved Arg residues in the arm structure of the C-terminal half of the HU-1 molecule and a Phe residue in the short beta-sheet connecting the two halves of the molecule are important for the DNA-binding ability and biological functions of this protein.
Goshima
N
N
Department of Molecular Genetics, Kyoto Pharmaceutical University, Japan.
Kohno
K
K
Imamoto
F
F
Kano
Y
Y
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D004269:DNA, Bacterial / Q000235:genetics* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D009154:Mutation; D014170:Transformation, Genetic
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; C040330:histone-like protein HU, bacteria