Identification and characterization of a novel RNA binding protein that associates with the 5'-untranslated region of the chloroplast psbA mRNA.
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.
Barnes
Dwight
D
Department of Cell Biology and Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA.
Cohen
Amybeth
A
Bruick
Richard K
RK
Kantardjieff
Katherine
K
Fowler
Sean
S
Efuet
Ekem
E
Mayfield
Stephen P
SP
D020121:5' Untranslated Regions; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002455:Cell Division; D002467:Cell Nucleus / Q000378:metabolism; D016825:Chlamydomonas reinhardtii / Q000378:metabolism; D002736:Chloroplasts / Q000378:metabolism*; D002942:Circular Dichroism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D019992:Databases as Topic; D004591:Electrophoresis, Polyacrylamide Gel; D015151:Immunoblotting; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D045332:Photosystem II Protein Complex / Q000378:metabolism*; D011485:Protein Binding; D017433:Protein Structure, Secondary; D012313:RNA / Q000737:chemistry / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000502:physiology*; D017386:Sequence Homology, Amino Acid; D013997:Time Factors
D020121:5' Untranslated Regions; D018076:DNA, Complementary; D045332:Photosystem II Protein Complex; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; C422369:photosystem II, psbA subunit; D012313:RNA