Reciprocal Regulation of Protein Synthesis and Carbon Metabolism for Thylakoid Membrane Biogenesis
(A) UV cross-linking experiment. A total of 200 ng of purified His-DLA2 were analyzed by UV cross-linking using 50–100 kcpm of radiolabeled psbA-, psbD, or rbcL- 5′ UTR RNA probes. (B) Competition experiments. We incubated 10 ng of His-DLA2 protein with radiolabeled psbA-RNA (psbA*) and a 5-, 25-, or 200-fold molar excess of the indicated competitor RNAs representing the 5′ UTRs of the psbA or rbcL mRNA, respectively. To exclude an unspecific RNA binding of contaminating E. coli proteins, we used the same volumes as used for the DLA2 protein of an elution fraction obtained from the bacterial host strain transformed with the empty expression vector served as control (E. coli protein, lane 3). (C) Equilibrium binding constant (KD) of recombinant DLA2 protein. Binding reactions containing 6.7 pM 32P-labeled psbA 5′ UTR RNA and indicated molarities of DLA2 were filtered through stacked nitrocellulose and nylon membranes (upper panel) using a dot blot apparatus according to Ostersetzer et al. . Signal intensities of nitrocellulose-bound protein–RNA complexes (Bound) as well as nylon membrane-bound free RNAs (Free) were quantified. The KD value was determined from three experiments performed with the same DLA2 preparation (lower panel).