Amino acid substitution in the C-terminal arm domain of HU-2 results in an enhanced affinity for DNA.
Three mutants of the Escherichia coli hupA gene, encoding the HU-2 protein, were constructed by synthetic oligodeoxyribonucleotide-directed, site-specific mutagenesis on M13mp18 vectors. The resulting HupAN10, HupAN11 and HupAN12 proteins contained Thr59-->Lys, Gln64-->Lys and Asn53-->Arg substitutions, respectively. These amino acid (aa) changes increased the positive charge of the N-terminal half of the two-strand, antiparallel beta-ribbon of the arm structure, which is believed to be a domain for DNA binding. The three mutant proteins bound to DNA more tightly than wild-type HU-2, and their affinities for DNA increased in the order of HupAN10, HupAN11, HupAN12. The mutant proteins showed a slightly increased HU activity for supporting Mu phage development. A mutant HU-2 protein with increased basicity, but with an altered aa sequence in the arm region due to a frameshift mutation, was also constructed. This mutant protein showed a reduced affinity to DNA and was unable to support Mu growth, suggesting that a unique aa sequence of the arm domain, rather than mere basicity of this domain, is required for efficient binding to DNA.
Goshima
N
N
Department of Molecular Genetics, Kyoto Pharmaceutical University, Japan.
Kano
Y
Y
Tanaka
H
H
Tanaka
H
H
Kohno
K
K
Yasuzawa
K
K
Imamoto
F
F
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D010583:Bacteriophage mu / Q000254:growth & development; D001483:Base Sequence; D015153:Blotting, Western; D004269:DNA, Bacterial / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D011485:Protein Binding
D000596:Amino Acids; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; C040330:histone-like protein HU, bacteria