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. 2011 Apr 12;23(4):1391–1403. doi: 10.1105/tpc.110.081950

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© 2011 American Society of Plant Biologists

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Figure 4. — BAM7 and BAM8 Proteins Bind a Specific Cis-Regulatory Element and Activate Gene Expression.

(A) EMSA using DIG-labeled oligonucleotides obtained by sequential rounds of RBSS. Oligonucleotides were incubated with recombinant BZR1-domains of BAM7 and BAM8. Input and eluates from rounds 4 to 7, and the final round 7 eluate of the control experiment (C), are shown. Full-length BAM7 and BAM8 (FL) bind to the most enriched oligonucleotide (EO). Single and double asterisks mark positions of unbound and bound oligonucleotides, respectively.

(B) The conserved BBRE motif isolated by RBSS contains known binding sites for transcriptional regulators including the BR-responsive element (BRRE) and the palindromic G box. The percentage value given below the residues indicates the conservation at this location in the motif from an alignment of 1,072,225 Illumina sequence reads. The asterisk indicates the base that was mutated to a thymine in the mBBRE.

(C) BZR1-domains and full-length BZR1-BAM proteins bind the BBRE but a single base mutation in the mBBRE is able to abolish the binding. Comp., a 300-fold excess of unlabeled BBRE (c) reduces binding, but a 300-fold excess of unlabeled mBBRE (c_m) does not. Only the location of the shifted oligonucleotides is shown. Note that lanes 5 to 7 were exposed for a shorter time than the rest of the EMSA. Input, dotblot of DIG-labeled oligonucleotides, shows equal labeling of the BBRE and the mBBRE (dilutions are indicated on the right).

(D) BAM8 activates reporter gene expression via the BBRE. Arabidopsis protoplasts were transfected with the Luc reporter under the control of the minimal CaMV 35S promoter (min35S), or with one (1xBBRE) or three copies of the BBRE sequence (3xBBRE) upstream of the min35S, or with three copies of mBBRE (3xmBBRE) upstream of the min35S. HA-tagged, full-length BAM7 or BAM8 served as effectors, and salmon sperm DNA was used as control DNA. Reporter activity is relative to the transfection control GUS and normalized to the average value obtained with the min35S using control DNA. Values are the mean LUC/GUS ratios of three replicate transformations performed in parallel (±sd).