In vitro mutagenesis of binding site elements for the clock-controlled proteins CCTR and Chlamy 1

The luciferin-binding protein (LBP) from the dinoflagellate, Gonyaulax polyedra, is regulated by a circadian clock at the translational level. A 22-nucleotide long interval in the lbp 3' untranslated region, which contains seven UG-repeats, was characterized as a circadian cis-acting element, to which a clock controlled factor (CCTR) binds. Recently we have found that the phylogenetically distant green alga, Chlamydomonas reinhardtii, contains a CCTR analog, called Chlamy 1. Here we show that the flanking nucleotides surrounding the UG-repeats are required for high binding activity of CCTR and Chlamy 1. The absence of three or more UG-repeats abolishes binding with both proteins.