CHARACTERIZATION OF THE SPERMIDINE-DEPENDENT, SEQUENCE-SPECIFIC ENDORIBONUCLEASE THAT REQUIRES TRANSFER-RNA FOR ITS ACTIVITY
CHARACTERIZATION OF THE SPERMIDINE-DEPENDENT, SEQUENCE-SPECIFIC ENDORIBONUCLEASE THAT REQUIRES TRANSFER-RNA FOR ITS ACTIVITY
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DOI：
10.1093/nar/20.14.3737
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发表时间：
1992-07-25
影响因子：
14.9
通讯作者：
NASHIMOTO, M
中科院分区：
文献类型：
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作者：
NASHIMOTO, M
小鼠 FM3A 细胞中的亚精胺依赖性序列特异性核糖核酸内切酶 (RNase 65) 由蛋白质和缺乏 3' 末端的转移 RNA 组成。使用部分纯化的酶来表征该酶的体外特性。 RNase 65 活性需要亚精胺，而精胺或 Mg++ 无法替代亚精胺。该酶可裂解磷酸二酯键 3' 侧的 RNA 底物。裂解反应的最佳温度约为 50 摄氏度，最佳 pH 值约为 7.0。该活性的最佳 KCl 浓度约为 10 mM。在 37 摄氏度和 50 摄氏度下分析了具有共同靶序列的两种不同折叠 RNA 底物的相对切割效率。该分析结果表明，靶序列的解折叠对于 RNase 65 的识别至关重要。此外，在使用几种旨在形成与野生型基本相同的二级结构的点突变 RNA 底物的实验中，靶序列中的 1 到 3 个核苷酸取代都会降低切割效率。 RNase 65 活性仅存在于细胞质提取物中，而不存在于细胞核提取物中。凝胶过滤分析表明核糖核酸内切酶的天然大小约为 150 kDa。
The spermidine-dependent, sequence-specific endoribonuclease (RNase 65) in mouse FM3A cells consists of protein and transfer RNA lacking its 3' terminus. In vitro properties of this enzyme were characterized using partially purified enzyme. The RNase 65 activity requires spermidine, which is not replaceable with spermine or Mg++. The enzyme cleaves an RNA substrate on the 3' side of the phosphodiester bond. The cleavage reaction has a temperature optimum around 50-degrees-C and a pH optimum around 7.0. The optimum KCl concentration for the activity is around 10 mM. Relative cleavage efficiency of two differently folded RNA substrates with the common target sequence was analyzed at 37-degrees-C and 50-degrees-C. The results of this analysis suggest that unfolding of the target sequence is critical for recognition by RNase 65. Furthermore, in experiments using several point-mutated RNA substrates designed to form basically the same secondary structure as the wild type, one to three nucleotide substitutions in the target sequence all reduced cleavage efficiency. The RNase 65 activity is found only in cytosolic extracts, not in nuclear ones. Gel filtration analysis suggests that the native size of the endoribonuclease is approximately 150 kDa.