Bernstein, D., Hook, B., Hajarnavis, A., Opperman, L. and Wickens, M.
Notes: PUF proteins act to reduce expression upon binding to the 3´UTR of target mRNA sequences. In this study, the binding of various PUF proteins (including C. elegans FBF-1) to known PUF binding sites was evaluated using a yeast three-hybrid system. For this assay, various PUF proteins fused to the Gal4 activation domain were expressed in pACT and pACT2 plasmids, and DNA oligos expressing PUF binding RNA sequences were cloned into the plasmid pIIIA/MS2-2. Assays were then performed in the yeast strain YBZ-1. β-galactosidase expression, indicating interaction between each PUF protein and the various binding sites, was measured using both solid- (colony lift) and liquid-phase assays. For the liquid-phase assays, β-galactosidase expression was measured using the Beta-Glo® Assay System. Cells were grown in selective media to an O.D.600 of 0.1–0.3, then mixed with an equal volume of Beta-Glo® Assay Reagent. After one hour, luminescence was measured. Luminescence values were normalized to cell number. The results of these binding assays were used to formulate a consensus PUF binding sequence, which was then used to screen a C. elegans 3´ UTR database for potential FBF-1 target sequences. (3458)