The Regulator of Nitrate Assimilation in Ascomycetes Is a Dimer Which Binds a Nonrepeated, Asymmetrical Sequence

ABSTRACT

The regulation of nitrate assimilation seems to follow the same pattern in all ascomycetes where this process has been studied. We show here by in vitro binding studies and a number of protection and interference techniques that the transcription factor mediating nitrate induction in Aspergillus nidulans, a protein containing a binuclear zinc cluster DNA binding domain, recognizes an asymmetrical sequence of the form CTCCGHGG. We further show that the protein binds to its consensus site as a dimer. We establish the role of the putative dimerization element by its ability to replace the analogous element of the cI protein of phage λ. Mutagenesis of crucial leucines of the dimerization element affect both the binding ability of the dimer and the conformation of the resulting protein-DNA complex. This is the first case to be described where a dimer recognizes such an asymmetrical nonrepeated sequence, presumably by each monomeric subunit making different contacts with different DNA half-sites.

ACKNOWLEDGMENTS

Joseph Strauss and M. Isabel Muro-Pastor contributed equally to this work.

We thank F. Gigliani for CSH50 E. coli cells, plasmid pC132, and λ phages used in this work; G. Cesareni for plasmid pC135; and F. Lema for polyclonal antibodies against the NirA(1–125) protein. J.S. is grateful to C. P. Kubicek, Technical University, and to the Department of Medical Biochemistry, University of Vienna, for laboratory space. Wilhelm Guschlbauer is thanked for helpful discussion.

This work was supported by CEE grants SCI-CT92-0815 and BIO2-CT93-0147. The work at Vienna was supported by grant 6105 from the Jubilaeumsfonds der Oesterreichischen Nationalbank to J.S. J.S. was supported by a studentship of Austauschprogramm France-Austria and by a short-term EMBO fellowship (ASTF 7958). M.I.M.-P. has been the recipient of, successively, a postdoctoral fellowship from the Ministerio de Educación y Ciencia of the Spanish Government, CE fellowship BIO-CT-94-8102, and a fellowship from Fondation pour la Recherche Médicale.