The active site of the thioredoxin-like domain of chloroplast protein disulfide isomerase, RB60, catalyzes the redox-regulated binding of chloroplast poly(A)-binding protein, RB47, to the 5' untranslated region of psbA mRNA.
RB60, a chloroplast protein disulfide isomerase, modulates the binding of RB47, chloroplast poly(A)-binding protein, to the 5'-UTR of the psbA mRNA using redox potential, allowing for a reversible switch capable of regulating psbA mRNA translation in a light/dark dependent manner. RB60 contains two thioredoxin-like domains with putative catalytic sites of -Cys-Gly-His-Cys- that are presumed to function as active sites for the redox-regulated changes in RNA-binding activity of RB47. To investigate whether these motifs are required for redox-regulated RNA binding, RNA-gel-mobility shift assays were performed with RB47 and mutant RB60 proteins with single cysteines changed to serines in the -Cys-Gly-His-Cys- motif. The results showed that each thioredoxin-like domain has independent catalytic function in the reactivation of RB47 binding and that a double active site mutant completely lacks the ability to activate RB47 RNA binding activity.
Kim
Jungmook
J
Department of Cell Biology and the Skaggs Institute for Chemical Biology, the Scripps Research Institute, 10550 North Torrey Pines Rd., La Jolla, CA 92037, U.S.A. jmkim@ksc.kumho.co.kr
Mayfield
Stephen P
SP
D020121:5' Untranslated Regions / Q000235:genetics / Q000378:metabolism; D020418:Algal Proteins / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D016825:Chlamydomonas reinhardtii / Q000201:enzymology / Q000235:genetics; D002736:Chloroplasts / Q000201:enzymology*; D024202:Electrophoretic Mobility Shift Assay / Q000379:methods; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D039102:Poly(A)-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D019704:Protein Disulfide-Isomerases / Q000235:genetics / Q000378:metabolism*; D018747:RNA, Chloroplast / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013879:Thioredoxins / Q000378:metabolism*
D020121:5' Untranslated Regions; D020418:Algal Proteins; D039102:Poly(A)-Binding Proteins; D018747:RNA, Chloroplast; D013879:Thioredoxins; D019704:Protein Disulfide-Isomerases