Analysis of the spacing between the two palindromes of activation sequence-1 with respect to binding to different TGA factors and transcriptional activation potential.
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 (approximately 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.
Krawczyk
Stefanie
S
Albrecht-von-Haller-Institut fuer Pflanzenwissenschaften, Universitaet Goettingen, Untere Karspuele 2, 37073 Goettingen, Germany.
Thurow
Corinna
C
Niggeweg
Ricarda
R
Gatz
Christiane
C
D001483:Base Sequence; D050976:Basic-Leucine Zipper Transcription Factors; D001665:Binding Sites; D004279:DNA, Viral / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D015967:Gene Expression Regulation, Viral; D017930:Genes, Reporter; D009687:Nuclear Proteins; D010940:Plant Proteins / Q000737:chemistry / Q000378:metabolism; D010942:Plant Viruses / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D020218:Response Elements / Q000235:genetics*; D016415:Sequence Alignment; D017384:Sequence Deletion / Q000235:genetics; D013379:Substrate Specificity; D013816:Thermodynamics; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism* / Q000821:virology; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism*; D015533:Transcriptional Activation
D050976:Basic-Leucine Zipper Transcription Factors; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D010940:Plant Proteins; C065794:TGA1a protein, Nicotiana tabacum; D014157:Transcription Factors; C068757:HBP-1b protein, plant