Identification and characterization of the RNA binding surface of the pentatricopeptide repeat protein

Keiko Kobayashi, Masuyo Kawabata, Keizo Hisano, Tomohiko Kazama, Ken Matsuoka, Mamoru Sugita, Takahiro Nakamura

Research output: Contribution to journalArticle

36 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)2712-2723
Number of pages12
JournalNucleic Acids Research
Volume40
Issue number6
DOIs
Publication statusPublished - Mar 1 2012

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RNA
Amino Acid Motifs
Proteins
Amino Acids
SELEX Aptamer Technique
Chloroplast Genes
Informatics
Nuclear Proteins
Mutagenesis
Organelles
Mitochondria
Genes

All Science Journal Classification (ASJC) codes

  • Genetics

Cite this

Identification and characterization of the RNA binding surface of the pentatricopeptide repeat protein. / Kobayashi, Keiko; Kawabata, Masuyo; Hisano, Keizo; Kazama, Tomohiko; Matsuoka, Ken; Sugita, Mamoru; Nakamura, Takahiro.

In: Nucleic Acids Research, Vol. 40, No. 6, 01.03.2012, p. 2712-2723.

Research output: Contribution to journalArticle

Kobayashi, Keiko ; Kawabata, Masuyo ; Hisano, Keizo ; Kazama, Tomohiko ; Matsuoka, Ken ; Sugita, Mamoru ; Nakamura, Takahiro. / Identification and characterization of the RNA binding surface of the pentatricopeptide repeat protein. In: Nucleic Acids Research. 2012 ; Vol. 40, No. 6. pp. 2712-2723.
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