Site-directed mutagenesis of the A-factor receptor protein: Val-41 important for DNA-binding and Trp-119 important for ligand-binding.
The A-factor receptor protein (ArpA) plays a key role in the regulation of secondary metabolism and cellular differentiation in Streptomyces griseus. ArpA binds the target DNA site forming a 22 bp palindrome in the absence of A-factor, and exogenous addition of A-factor to the ArpA-DNA complex immediately releases ArpA from the DNA. An amino acid (aa) replacement at Val-41 to Ala in an alpha-helix-turn-alpha-helix (HTH) motif at the N-terminal portion of ArpA abolished DNA-binding activity but not A-factor-binding activity, suggesting the involvement of this HTH in DNA-binding. On the other hand, an aa replacement at Trp-119 to Ala generated a mutant ArpA that was unable to bind A-factor, thus resulting in an A-factor-insensitive mutant that bound normally to its target DNA in both the presence and absence of A-factor. These data suggest that ArpA consisting of two functional domains, one for HTH-type DNA-binding at the N-terminal portion and one for A-factor-binding at the C-terminal portion, is a member of the LacI family. Consistent with this, two ArpA homologues, CprA and CprB, from Streptomyces coelicolor A3(2), each of which contains a very similar aa sequence of the HTH to that of ArpA, also recognized and bound the same DNA target. However, neither CprA nor CprB recognized A-factor, probably due to much less similarity in the C-terminal domains.
Sugiyama
M
M
Department of Biotechnology, Graduate School of Agriculture and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan.
Onaka
H
H
Nakagawa
T
T
Horinouchi
S
S
D015107:4-Butyrolactone / Q000031:analogs & derivatives* / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001665:Binding Sites; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D006133:Growth Substances / Q000378:metabolism*; D008024:Ligands; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D013302:Streptomyces; D013305:Streptomyces griseus
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D006133:Growth Substances; D008024:Ligands; D012097:Repressor Proteins; C025035:A-factor (Streptomyces); D015107:4-Butyrolactone