Disulfide bond formation between RNA binding domains is used to regulate mRNA binding activity of the chloroplast poly(A)-binding protein.
Binding of the chloroplast poly(A)-binding protein, RB47, to the psbA mRNA is regulated in response to light and is required for translation of this mRNA in chloroplasts. The RNA binding activity of RB47 can be modulated in vitro by oxidation and reduction. Site-directed mutations to individual cysteine residues in each of the four RNA binding domains of RB47 showed that changing single cysteines to serines in domains 2 or 3 reduced, but did not eliminate, the ability of RB47 to be redox-regulated. Simultaneously changing cysteines to serines in both domains 2 and 3 resulted in the production of RB47 protein that was insensitive to redox regulation but retained the ability to bind the psbA mRNA at high affinity. The poly(A)-binding protein from Saccharomyces cerevisiae lacks cysteine residues in RNA binding domains 2 and 3, and this poly(A)-binding protein lacks the ability to be regulated by oxidation or reduction. These data show that disulfide bond formation between RNA binding domains in a poly(A)-binding protein can be used to regulate the ability of this protein to bind mRNA and suggest that redox regulation of RNA binding activity may be used to regulate translation in organisms whose poly(A)-binding proteins contain these critical cysteine residues.
Fong
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Department of Cell Biology and the Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California 92037, USA.
Lentz
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Mayfield
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D000595:Amino Acid Sequence; D002736:Chloroplasts / Q000378:metabolism*; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D003553:Cystine / Q000235:genetics / Q000378:metabolism*; D018506:Gene Expression Regulation, Plant; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D045322:Photosynthetic Reaction Center Complex Proteins / Q000235:genetics*; D045332:Photosystem II Protein Complex; D039102:Poly(A)-Binding Proteins; D011485:Protein Binding; D014176:Protein Biosynthesis; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000378:metabolism*; D018749:RNA, Plant / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
D045322:Photosynthetic Reaction Center Complex Proteins; D045332:Photosystem II Protein Complex; D039102:Poly(A)-Binding Proteins; D012333:RNA, Messenger; D018749:RNA, Plant; D016601:RNA-Binding Proteins; D003553:Cystine; D003545:Cysteine