Chimeric HU-IHF proteins that alter DNA-binding ability.
Chimeric proteins between Escherichia coli histone-like HU and IHF were constructed by genetic engineering, in which part of the arm region was replaced by the corresponding region of IHF alpha (designated as HupANhimA) or IHF beta (HupANhimD); alternatively, an alpha-helix 2-beta 1 region was replaced by the corresponding region of IHF alpha (HupAXhimA) or IHF beta (HupAXhimD) (symbols N and X indicate NotI and XhoI junctions). These proteins were synthesized in a hupA-hupB double-deletion mutant. HupANhimA exhibited marked reduction in nonspecific DNA binding in vitro, and a drastic loss of HU activity in replicative transposition of Mu phage in vivo. HupANhimD also showed a significant reduction in the ability for DNA binding, though this protein supported Mu phage development. In contrast, the other two chimeric HU proteins showed only slight changes in nonspecific DNA-binding ability: they retained activities for transposition of Mu phage in vivo. These observations confirm that the flexible arm of HU-2, a domain proposed for DNA binding [Tanaka et al., Nature 310 (1984) 376-381; Goshima et al., Gene 96 (1990) 141-145], plays an important role in the physiological function of this protein. The results indicate that a unique conformation of the arm structure of HU protein, particularly the N-terminal half of a two-strand antiparallel beta-ribbon of the structure, is important for the DNA-binding ability of this protein.
Goshima
N
N
Department of Molecular Genetics, Kyoto Pharmaceutical University, Japan.
Inagaki
Y
Y
Otaki
H
H
Tanaka
H
H
Hayashi
N
N
Imamoto
F
F
Kano
Y
Y
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D010583:Bacteriophage mu / Q000254:growth & development; D001483:Base Sequence; D004252:DNA Mutational Analysis; D004269:DNA, Bacterial / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics*; D034982:Integration Host Factors; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D014779:Virus Replication
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D034982:Integration Host Factors; D011993:Recombinant Fusion Proteins; C040330:histone-like protein HU, bacteria