Hande S, Jayabaskaran C
Department of Biochemistry, Indian Institute of Science, Bangalore, India.
Biochem Mol Biol Int. 1998 Feb;44(2):417-24. doi: 10.1080/15216549800201432.
To study the sequential steps in the processing pathway of the chloroplast monocistronic intronless tRNA precursors, we examined cucumber chloroplast tRNA(Leu)(CAA) processing in a cucumber or pea chloroplast soluble extract. The tRNA(Leu)(CAA) precursor synthesized from SP6 RNA polymerase-directed transcription system, was used as a substrate. Incubation of the tRNA precursor with the pea extract resulted in processing of tRNA(Leu)(CAA) via 5'- and 3'-endonucleolytic cleavages followed by final trimming of extra 3' nucleotides by 3' exonuclease(s). No preferred order for endonucleolytic cleavages has been observed during the in vitro tRNA(Leu) processing and the simultaneous occurrence of the intermediates consisting of leader + tRNA(Leu) and tRNA(Leu) + trailer, indicate that either 5'- or 3'-endonucleolytic cleavage can occur as the first step in vitro.
为了研究叶绿体单顺反子无内含子tRNA前体加工途径中的连续步骤，我们在黄瓜或豌豆叶绿体可溶性提取物中检测了黄瓜叶绿体tRNA(Leu)(CAA)的加工过程。由SP6 RNA聚合酶指导转录系统合成的tRNA(Leu)(CAA)前体用作底物。将tRNA前体与豌豆提取物一起温育，导致tRNA(Leu)(CAA)通过5'-和3'-内切核酸酶切割进行加工，随后由3'-外切核酸酶对额外的3'核苷酸进行最终修剪。在体外tRNA(Leu)加工过程中未观察到内切核酸酶切割的优先顺序，并且由前导序列+tRNA(Leu)和tRNA(Leu)+尾随序列组成的中间体同时出现，表明5'-或3'-内切核酸酶切割都可以作为体外的第一步发生。
