Identification and Characterization of a Novel RNA Binding Protein That Associates with the 5′-Untranslated Region of the Chloroplast psbA mRNA ² Dwight Barnes, ‡,§ Amybeth Cohen,* ,‡,| Richard K. Bruick, §,⊥ Katherine Kantardjieff, # Sean Fowler, | Ekem Efuet, §,4 and Stephen P. Mayfield § Department of Biological Science, Department of Chemistry and Biochemistry, and W. M. Keck Foundation Center for Molecular Structure, California State UniVersity Fullerton, Fullerton, California 92834-6850, and Department of Cell Biology and Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037 ReceiVed October 27, 2003; ReVised Manuscript ReceiVed March 17, 2004 ABSTRACT: Binding of proteins to chloroplast-encoded mRNAs has been shown to be an essential part of chloroplast gene expression. Four nuclear-encoded proteins (38, 47, 55, and 60 kDa) have been identified that bind to the 5′-untranslated region of the Chlamydomonas reinhardtii psbA mRNA with high affinity and specificity. We have cloned a cDNA that represents the 38 kDa protein (RB38) and show that it encodes a novel RNA binding protein that is primarily localized within the chloroplast stroma. RB38 contains four 70 amino acid repeats with a high percentage of basic amino acids, as well as an amino- terminal extension predicted to act as a chloroplast import sequence. We demonstrate that the 38 kDa precursor protein is imported into isolated chloroplasts and interacts with high specificity to uridine-rich regions within the 5′-untranslated region of the psbA mRNA. While database searches have identified hypothetical proteins from several other eukaryotic species with high sequence similarity to the deduced amino acid sequence of RB38, no proteins with homology to RB38 have been biochemically characterized. Bioinformatic analysis of the RB38 sequence, together with structure analysis using circular dichroism and protein modeling, suggests that the 70 amino acid repeats within RB38 are similar in fold to previously identified RNA binding motifs, despite limited sequence homology. Regulation of eukaryotic gene expression involves a number of posttranscriptional processes, including those affecting mRNA stability and translation. The interaction between RNA binding proteins and the corresponding RNA elements found within particular mRNAs has been shown to be an essential part of translational regulation. Conserved themes emerge among a variety of organisms when evaluat- ing mRNA binding proteins and the RNA elements they recognize. The expanding database of RNA structures and protein complexes suggests that unique protein-RNA in- teractions are based on a relatively small set of functional groups (1), and these interactions can have profound effects on the expression of specific genes. The 5′- and 3′-untranslated regions (UTRs) 1 of both prokaryotic and eukaryotic mRNAs play important roles in translation. Often, these RNA elements are bound by specific proteins as a means to regulate the translation of the adjacent coding region. For example, poly(A) binding protein (PABP) has been shown to play an important role in the initiation of eukaryotic protein synthesis through an interaction with eIF4G (yeast and plants) and eIF4B (plants) at the 5′ end of the mRNA and direct interaction with the poly(A) tail on the 3′ end of the mRNA (2-4). Prokaryotic systems also utilize mRNA binding proteins for translational regulation. However, these proteins typically inhibit translation after binding to the 5′-UTR (5). Thus, RNA binding protein interaction with specific RNA elements within UTRs is a universal mechanism to enhance or inhibit translation. The majority of mRNA binding proteins described to date contain  sheets that act to stabilize the interaction of charged and aromatic side chains of the proteins with the RNA (1). Most of these proteins contain a combination of  sheets and R helices that form the well-characterized RNA recogni- tion motif (RRM) that binds to target RNA. PABP contains a total of four RRMs that are positioned within conserved amino acid sequences. Deo et al. (6) have shown by X-ray ² This study was supported by funds from the Department of Energy (ER15313) and the National Institutes of Health (GM54659) to S.P.M. and by NIH Grant GM58808-01 to A.C. E.E. was supported by a Skaggs postdoctoral fellowship. * To whom correspondence should be addressed. E-mail: acohen@ fullerton.edu. Fax: (714) 278-3426. Phone: (714) 278-2178. ‡ D.B. and A.C. contributed equally to this work. § The Scripps Research Institute. | Department of Biological Science, California State University Fullerton. ⊥ Present address: Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX 75390-9152. # Department of Chemistry and Biochemistry and W. M. Keck Foundation Center for Molecular Structure, California State University Fullerton. 4 Present address: The University of Texas M. D. Anderson Cancer Center, Houston, TX 77030. 1 Abbreviations: UTR, untranslated region; PABP, poly(A) binding protein; RRM, RNA recognition motif; KH, K homology; RBD, RNA binding domain; RBS, ribosome binding site; PDI, protein disulfide isomerase; wt, wild type; IVT, in vitro translation; PAGE, polyacryl- amide gel electrophoresis; PDB, Protein Data Bank. 8541 Biochemistry 2004, 43, 8541-8550 10.1021/bi035909j CCC: $27.50 © 2004 American Chemical Society Published on Web 06/12/2004