Nucleic Acids Research, 2002, Vol. 30, No. 3 775-781
© 2002 Oxford University Press
Analysis of the spacing between the two palindromes of activation sequence-1 with respect to binding to different TGA factors and transcriptional activation potential
Albrecht-von-Haller-Institut fuer Pflanzenwissenschaften, Universitaet Goettingen, Untere Karspuele 2, 37073 Goettingen, Germany
In higher plants, activation sequence-1 (as-1) of the cauliflower mosaic virus 35S promoter mediates both salicylic acid- and auxin-inducible transcriptional activation. Originally found in viral and T-DNA promoters, as-1-like elements are also functional elements of plant promoters activated in the course of a defence response upon pathogen attack. as-1-like elements are characterised by two imperfect palindromes with the palindromic centres being spaced by 12 bp. They are recognised by plant nuclear as-1-binding factor ASF-1, the major component of which is basic/leucine zipper (bZIP) protein TGA2.2 (
80%) in Nicotiana tabacum. In electrophoretic mobility shift assays, ASF-1 as well as bZIP proteins TGA2.2, TGA2.1 and TGA1a showed a 3–10-fold reduced binding affinity to mutant as-1 elements encoding insertions of 2, 4, 6, 8 or 10 bp between the palindromes, respectively. This correlated with a 5–10-fold reduction in transcriptional activation from these elements in transient expression assays. Although ASF-1 and TGA factors bound efficiently to a mutant element carrying a 2 bp deletion between the palindromes [as-1/(–2)], the latter was strongly compromised with respect to mediating gene expression in vivo. A fusion protein consisting of TGA2.2 and a constitutive activation domain mediated transactivation from as-1/(–2) demonstrating binding of TGA factors in vivo. We therefore conclude that both DNA binding and transactivation require optimal positioning of TGA factors on the as-1 element.
* To whom correspondence should be addressed. Tel: +49 551 39 7843; Fax: +49 551 39 7820; Email: cgatz{at}gwdg.de Present address: Ricarda Niggeweg, John Innes Centre, Norwich NR4 7UH, UK
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