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 redoxregulated. 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.Translational regulation is the predominant mechanism for controlling gene expression in the chloroplast of plants and algae (1-3). In Chlamydomonas reinhardtii, translational regulation of the chloroplast psbA gene, which encodes the D1 protein (a major component of photosystem II), appears to require the binding of a complex composed of four nuclearencoded proteins (4). The 47-kDa member of this protein complex (RB47) has been identified as a polyadenylate-binding protein (PABP) 1 (5). Physiological studies have shown that both RB47 RNA binding activity and psbA translation are activated in a light-dependent manner (4). Characterization of nuclear mutants lacking RB47 has shown that this protein is required for binding of the complex to the 5Ј-untranslated region (UTR) of the psbA mRNA (6). Absence of RB47 results in the failure of the psbA mRNA to associate with polyribosomes, and hence the loss of translation, suggesting that RB47 acts as a message-specific translation initiation factor for the psbA mRNA (6). Binding of the RB47 protein to the 5Ј-UTR of the psbA mRNA can be modulated in vitro by oxidation and reduction reactions (7). The translational machinery of the chloroplast has similarities to prokaryotes including 70 S ribosomes and mRNAs that lack poly(A) tails and 7-methyl-G caps. PABPs have not as yet been identified in prokaryotic organisms. However, the 5Ј-UTR of the psbA mRNA is A/U-rich and contains two stretches of A residues that have been identified as the primary binding site for the RB47 proteins (8). RB47 contains the four RNA recognition motifs (RRMs), which are highly conserved in all PABPs. The structure of RRM1 and RRM2 of the human PABP has been determined (9) and shows a ␤␣␤␤␣␤ secondary structure for each of the RNA binding domain...