Ostheimer Gerard J, Rojas Margarita, Hadjivassiliou Haralambos, Barkan Alice
Institute of Molecular Biology, Department of Chemistry and Department of Biology, University of Oregon, Eugene, Oregon 97403, USA.
J Biol Chem. 2006 Feb 24;281(8):4732-8. doi: 10.1074/jbc.M508921200. Epub 2005 Dec 26.
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.
CRS2相关因子1和2（CAF1和CAF2）是密切相关的蛋白质，它们与叶绿体RNA剪接因子2（CRS2）协同作用，促进玉米叶绿体中特定II类内含子的剪接。CRS2-CAF复合物在体内与它们同源的II类内含子紧密结合，其中CAF亚基决定了复合物的内含子特异性。在这项研究中，我们发现CRS2-CAF复合物在没有内含子靶标的情况下是稳定的，并且CRS2结合CAF2羧基末端区域中一个22个氨基酸的基序，该基序在CAF1中是保守的。使用重组蛋白进行的酵母双杂交试验和共分级研究表明，该基序对于结合CRS2既必要又充分。预测该22个氨基酸的基序会形成一个两亲性螺旋，其疏水表面在CAF1和CAF2之间是保守的。我们提出，该表面结合CRS2表面上的疏水补丁，先前已证明该补丁对于CRS2与CAF2之间的相互作用是必需的。
