Sequence-specific binding of a chloroplast pentatricopeptide repeat protein to its native group II intron ligand.
Pentatricopeptide repeat (PPR) proteins are defined by degenerate 35-amino acid repeats that are related to the tetratricopeptide repeat (TPR). Most characterized PPR proteins mediate specific post-transcriptional steps in gene expression in mitochondria or chloroplasts. However, little is known about the structure of PPR proteins or the biochemical mechanisms through which they act. Here we establish features of PPR protein structure and nucleic acid binding activity through in vitro experiments with PPR5, which binds and stabilizes a chloroplast tRNA precursor harboring a group II intron. Recombinant PPR5 was shown to be monomeric by analytical ultracentrifugation and gel filtration. Circular dichroism spectroscopy showed that PPR5 has a high content of alpha helices, as predicted from the similarity between PPR and TPR motifs. PPR5 and another PPR protein, CRP1, bind with high affinity to single-stranded RNA, but bind poorly to single-stranded DNA or to double-stranded RNA or DNA. A specific PPR5 binding site was identified within its group II intron ligand. The minimal site spans approximately 45 nucleotides, encompasses two group II intron functional motifs, and overlaps the terminus of an in vivo RNA decay product. These results suggest mechanisms by which PPR5 influences both RNA stability and splicing.
Williams-Carrier
Rosalind
R
Department of Biology, Institute of Molecular Biology, University of Oregon, Eugene, Oregon 97403, USA.
Kroeger
Tiffany
T
Barkan
Alice
A
D001665:Binding Sites; D002736:Chloroplasts / Q000378:metabolism*; D004247:DNA / Q000378:metabolism*; D007438:Introns; D008024:Ligands; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D017433:Protein Structure, Secondary; D012313:RNA / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D020449:Repetitive Sequences, Amino Acid; D003313:Zea mays / Q000235:genetics / Q000378:metabolism*
D008024:Ligands; D010940:Plant Proteins; D011994:Recombinant Proteins; D012313:RNA; D004247:DNA