Reciprocal Regulation of Protein Synthesis and Carbon Metabolism for Thylakoid Membrane Biogenesis
Figure 11
Binding of RNA by dihydrolipoamide acetyltransferases might be a global phenomenon.
(A) Hexahistidine-tagged E2 fusion proteins from C. reinhardtii (Cr), S. cerevisiae (Sc), Synechocystis sp. 6803 (Syn), and H. sapiens (Hs) along with two control proteins (PratA and RBP40) were purified on Ni-NTA Sepharose, separated by SDS-PAGE, and Coomassie-stained. To exclude an unspecific RNA binding of contaminating E. coli proteins in (B), we used the same volumes as used for the C. reinhardtii E2 protein of an elution fraction obtained from the bacterial host strain transformed with the empty expression vector served as control (eV). Recombinant proteins are indicated by arrows. Proteins in the preparation of the human E2 subunit (Hs) that are specifically recognized by an anti-histidine antibody are marked by an asterisk. (B) RNA binding assay. One of the 20 (∼100 ng) recombinant proteins shown in (A) was used for UV cross-linking to psbA mRNA. Lanes “psbA*” and “psbA” show the radiolabeled psbA RNA without and with RNase treatment, respectively. Due to the high intensity of the psbA* signal, a lower exposure of this lane is shown. Specific radioactive signals are indicated by arrows.