Saldanha R, Chen B, Wank H, Matsuura M, Edwards J, Lambowitz A M
Institute for Cellular and Molecular Biology, Departments of Chemistry and Biochemistry, and Section of Molecular Genetics and Microbiology, School of Biological Sciences, University of Texas at Austin, Austin, Texas 78712, USA.
Biochemistry. 1999 Jul 13;38(28):9069-83. doi: 10.1021/bi982799l.
Group II introns encode proteins with reverse transcriptase activity. These proteins also promote RNA splicing (maturase activity) and then, with the excised intron, form a site-specific DNA endonuclease that promotes intron mobility by reverse splicing into DNA followed by target DNA-primed reverse transcription. Here, we used an Escherichia coli expression system for the Lactococcus lactis group II intron Ll.LtrB to show that the intron-encoded protein (LtrA) alone is sufficient for maturase activity, and that RNP particles containing only the LtrA protein and excised intron RNA have site-specific DNA endonuclease and target DNA-primed reverse transcriptase activity. Detailed analysis of the splicing reaction indicates that LtrA is an intron-specific splicing factor that binds to unspliced precursor RNA with a K(d) of </=0.12 pM at 30 degrees C. This binding occurs in a rapid bimolecular reaction, which is followed by a slower step, presumably an RNA conformational change, required for splicing to occur. Our results constitute the first biochemical analysis of protein-dependent splicing of a group II intron and demonstrate that a single intron-encoded protein can interact with the intron RNA to carry out a coordinated series of reactions leading to splicing and mobility.
II 组内含子编码具有逆转录酶活性的蛋白质。这些蛋白质还促进 RNA 剪接（成熟酶活性），然后与切除的内含子一起形成位点特异性 DNA 内切核酸酶，该酶通过反向剪接进入 DNA 并随后进行靶 DNA 引发的逆转录来促进内含子的移动。在这里，我们使用大肠杆菌表达系统来表达乳酸乳球菌的 II 组内含子 Ll.LtrB，结果表明仅内含子编码的蛋白质（LtrA）就足以具有成熟酶活性，并且仅包含 LtrA 蛋白和切除的内含子 RNA 的核糖核蛋白颗粒具有位点特异性 DNA 内切核酸酶和靶 DNA 引发的逆转录酶活性。对剪接反应的详细分析表明，LtrA 是一种内含子特异性剪接因子，在 30℃时以 K(d)≤0.12 pM 的亲和力与未剪接的前体 RNA 结合。这种结合发生在一个快速的双分子反应中，随后是一个较慢的步骤，大概是剪接发生所需的 RNA 构象变化。我们的结果构成了对 II 组内含子蛋白质依赖性剪接的首次生化分析，并证明单个内含子编码的蛋白质可以与内含子 RNA 相互作用，以进行导致剪接和移动的一系列协调反应。
