Figure 8

Interaction of the miR156 SBP-binding motif (GTAC core sequence) with the SBP protein domain of SPL15. (A) Consensus DNA sequence present in the promoter region of six previously reported genes interacting with SBP DNA binding domains. CCR1-bx represents the GTAC motif binding to SBP protein CCR1 from Chlamydomonas reinhardtii[28, 29]; SBP1/2-bx1 represents the GTAC motif binding to SBP1 and SBP2 in Antirrhinum majus[30]; SPL3-bx1 and -bx2 [5, 11], SPL7-bx [32]; and SPL14-bx [31] represents the GTAC motif binding to corresponding SPL proteins from Arabidopsis thaliana. Yellow boxes, 100% conserved; green boxes, 67-83% conserved. The degree of conservation is indicated in the schematic by the height of the letters (measured as bits). (B) GTAC core repeats present in the miR156b promoter sequence (from bp -1 to -1700). (C) SDS-PAGE analysis of His-tagged recombinant SBP peptide expressed in E. coli. Lane 1, protein marker; lane 2, protein extract of non-induced E. coli cells carrying the SBP domain in pET28a; lane 3, protein extract of induced E. coli carrying the SBP domain. (D) Western blot of resolved proteins in C using anti-His antibody. (E) Electrophoretic mobility shift assay illustrating His-tagged SBP peptide bound to a labeled miR156b promoter fragment containing tandem repeats of the GTAC core. Lane 1, labeled DNA plus SBP peptide. lane 2, labeled DNA only; lane 3, labeled DNA plus SBP protein and 100-fold excess unlabeled DNA. Black arrow shows the shifted band in lane 1.