Nucleic Acids Research, 2001, Vol. 29, No. 24 4973-4982
© 2001 Oxford University Press
The protein ORF80 from the acidophilic and thermophilic archaeon Sulfolobus islandicus binds highly site-specifically to double-stranded DNA and represents a novel type of basic leucine zipper protein
University Bayreuth, Biochemistry II, Universitätsstrasse 30, 95447 Bayreuth, Germany
The cryptic high copy number plasmid pRN1 from the thermophilic and acidophilic crenarchaeote Sulfolobus islandicus shares three conserved open reading frames with other S.islandicus plasmids. One of the open reading frames, namely orf80, encodes a 9.5 kDa protein that has no homology to any characterised protein. Recombinant ORF80 purified from Escherichia coli binds to double-stranded DNA in a sequence-specific manner as suggested by EMSA experiments and DNase I footprints. Two highly symmetrical binding sites separated by 
60 bp were found upstream of the orf80 gene. Both binding sites contain two TTAA motifs as well as other conserved bases. Fluorescence measurements show that short duplex DNAs derived from a single binding site sequence are bound with submicromolar affinity and moderate cooperativity by ORF80. On DNA fragments carrying both binding sites, a rather large protein–DNA complex is formed in a highly cooperative manner. ORF80 contains an N-terminal leucine zipper motif and a highly basic domain at its C-terminus. Compared to all known basic leucine zipper proteins the order of the domains is reversed in ORF80. ORF80 may therefore constitute a new subclass of basic leucine zipper DNA-binding proteins.
* To whom correspondence should be addressed. Tel: +49 921 55 2419; Fax: +49 921 55 2432; Email: georg.lipps{at}uni-bayreuth.de Present address:Pablo Ibanez, INSERM U289, Hôpital de la Pitie Salpétrière, 47 Boulevard de l’Hôpital, 75013 Paris, France
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