title: Identification and characterization of the RNA binding surface of the pentatricopeptide repeat protein.
abstract: The expressions of chloroplast and mitochondria genes are tightly controlled by numerous nuclear-encoded proteins, mainly at the post-transcriptional level. Recent analyses have identified a large, plant-specific family of pentatricopeptide repeat (PPR) motif-containing proteins that are exclusively involved in RNA metabolism of organelle genes via sequence-specific RNA binding. A tandem array of PPR motifs within the protein is believed to facilitate the RNA interaction, although little is known of the mechanism. Here, we describe the RNA interacting framework of a PPR protein, Arabidopsis HCF152. First, we demonstrated that a Pfam model could be relevant to the PPR motif function. A series of proteins with two PPR motifs showed significant differences in their RNA binding affinities, indicating functional differences among PPR motifs. Mutagenesis and informatics analysis putatively identified five amino acids organizing its RNA binding surface [the 1st, 4th, 8th, 12th and 'ii'(-2nd) amino acids] and their complex connections. SELEX (Systematic evolution of ligands by exponential enrichment) and nucleobase preference assays determined the nucleobases with high affinity for HCF152 and suggested several characteristic amino acids that may be involved in determining specificity and/or affinity of the PPR/RNA interaction.
authors: Faculty of Agriculture, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, Fukuoka 812-8581, Japan.
authors: Keiko
authors: K
authors: Kobayashi
authors: Masuyo
authors: M
authors: Kawabata
authors: Keizo
authors: K
authors: Hisano
authors: Tomohiko
authors: T
authors: Kazama
authors: Ken
authors: K
authors: Matsuoka
authors: Mamoru
authors: M
authors: Sugita
authors: Takahiro
authors: T
authors: Nakamura
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D056510:Position-Specific Scoring Matrices; D011485:Protein Binding; D012313:RNA / Q000737:chemistry* / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D000596:Amino Acids; D029681:Arabidopsis Proteins; C477515:HCF152 protein, Arabidopsis; D016601:RNA-Binding Proteins; D012313:RNA