Formation of the CRS2-CAF2 group II intron splicing complex is mediated by a 22-amino acid motif in the COOH-terminal region of CAF2.
CRS2-associated factors 1 and 2 (CAF1 and CAF2) are closely related proteins that function in concert with chloroplast RNA splicing 2 (CRS2) to promote the splicing of specific sets of group II introns in maize chloroplasts. The CRS2-CAF complexes bind tightly to their cognate group II introns in vivo, with the CAF subunit determining the intron specificity of the complex. In this work we show that the CRS2-CAF complexes are stable in the absence of their intron targets and that CRS2 binds a 22 amino acid motif in the COOH-terminal region of CAF2 that is conserved in CAF1. Yeast two-hybrid assays and co-fractionation studies using recombinant proteins show that this motif is both necessary and sufficient to bind CRS2. The 22-amino acid motif is predicted to form an amphipathic helix whose hydrophobic surface is conserved between CAF1 and CAF2. We propose that this surface binds the hydrophobic patch on the surface of CRS2 previously shown to be necessary for the interaction between CRS2 and CAF2.
Ostheimer
Gerard J
GJ
Institute of Molecular Biology, Department of Chemistry and Department of Biology, University of Oregon, Eugene, Oregon 97403, USA.
Rojas
Margarita
M
Hadjivassiliou
Haralambos
H
Barkan
Alice
A
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000378:metabolism*; D002265:Carboxylic Ester Hydrolases / Q000235:genetics*; D002736:Chloroplasts / Q000378:metabolism*; D007438:Introns; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016366:Open Reading Frames; D010940:Plant Proteins / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D012313:RNA / Q000737:chemistry; D012326:RNA Splicing; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D020798:Two-Hybrid System Techniques; D003313:Zea mays / Q000378:metabolism*
D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D012313:RNA; C423323:CRS2 protein, Zea mays; D002265:Carboxylic Ester Hydrolases