Ostheimer Gerard J, Hadjivassiliou Haralambos, Kloer Daniel P, Barkan Alice, Matthews Brian W
Institute of Molecular Biology, University of Oregon, Eugene, OR 97403, USA.
J Mol Biol. 2005 Jan 7;345(1):51-68. doi: 10.1016/j.jmb.2004.10.032.
Chloroplast RNA splicing 2 (CRS2) is a nuclear-encoded protein required for the splicing of nine group II introns in maize chloroplasts. CRS2 functions in the context of splicing complexes that include one of two CRS2-associated factors (CAF1 and CAF2). The CRS2-CAF1 and CRS2-CAF2 complexes are required for the splicing of different subsets of CRS2-dependent introns, and they bind tightly and specifically to their genetically defined intron targets in vivo. The CRS2 amino acid sequence is closely related to those of bacterial peptidyl-tRNA hydrolases (PTHs). To identify the structural differences between CRS2 and bacterial PTHs responsible for CRS2's gains of CAF binding and intron splicing functions, we determined the structure of CRS2 by X-ray crystallography. The fold of CRS2 is the same as that of Escherichia coli PTH, but CRS2 has two surfaces that differ from the corresponding surfaces in PTH. One of these is more hydrophobic in CRS2 than in PTH. Site-directed mutagenesis of this surface blocked CRS2-CAF complex formation, indicating that it is the CAF binding site. The CRS2 surface corresponding to the putative tRNA binding face of PTH is considerably more basic than in PTH, suggesting that CRS2 interacts with group II intron substrates via this surface. Both the sequence and the structural context of the amino acid residues essential for peptidyl-tRNA hydrolase activity are conserved in CRS2, yet expression of CRS2 is incapable of rescuing a pth(ts)E.coli strain.
叶绿体RNA剪接2（CRS2）是一种核编码蛋白，是玉米叶绿体中9个II类内含子剪接所必需的。CRS2在剪接复合体的背景下起作用，该复合体包括两种CRS2相关因子（CAF1和CAF2）之一。CRS2-CAF1和CRS2-CAF2复合体是CRS2依赖性内含子不同子集剪接所必需的，并且它们在体内紧密且特异性地结合其基因定义的内含子靶标。CRS2氨基酸序列与细菌肽基-tRNA水解酶（PTH）的序列密切相关。为了确定CRS2与负责CRS2获得CAF结合和内含子剪接功能的细菌PTH之间的结构差异，我们通过X射线晶体学确定了CRS2的结构。CRS2的折叠与大肠杆菌PTH相同，但CRS2有两个与PTH中相应表面不同的表面。其中一个在CRS2中比在PTH中更疏水。该表面的定点诱变阻止了CRS2-CAF复合体的形成，表明它是CAF结合位点。与PTH假定的tRNA结合面相对应的CRS2表面比PTH中的碱性强得多，这表明CRS2通过该表面与II类内含子底物相互作用。肽基-tRNA水解酶活性所必需的氨基酸残基的序列和结构背景在CRS2中都保守，但CRS2的表达无法拯救pth(ts)大肠杆菌菌株。
