Selection of a preribosomal RNA processing site by a nucleolar endoribonuclease involves formation of a stable complex.
Selection of a preribosomal RNA processing site by a nucleolar endoribonuclease involves formation of a stable complex.
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核仁核糖核酸内切酶对前核糖体 RNA 加工位点的选择涉及稳定复合物的形成。
DOI：
10.1093/nar/21.24.5775
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发表时间：
1993
影响因子：
14.9
通讯作者：
Shumard,CM
中科院分区：
文献类型：
--
作者：
Eichler,DC;Liberatore,JA;Shumard,CM
来自小鼠埃利希腹水肿瘤细胞的核核核糖核酸内切酶，与小鼠前体核糖体RNA的核内溶分裂有关，特异且稳定地结合含有+650早期加工位点的体外衍生rRNA转录物。通过迁移率转移分析、甘油梯度速度沉降分析和紫外交联研究证明了这种结合的特异性。结合不需要Mg2+，因此不依赖于裂解；然而，结合依赖于早期+650加工位点的存在，因为删除+650加工位点的pre-rRNA转录本无法竞争结合。小的核仁RNA成分不需要形成这种稳定的复合物，也不需要加工能力强的prerna转录物的特异性切割。使用32标记的5-叠氮吡啶取代的前rRNA与结合核核核糖核酸内切酶进行紫外交联研究，鉴定出三个大小接近的分别为ñ50、ñ48和ñ45 kDa的多肽，它们特异性地交联到加工能力强的rRNA转录物上。这三种多肽通过核糖核酸酶酶切和sdpolyacrylamide凝胶电泳鉴定。标记多肽的相同模式也从凝胶迁移转移分析中确定，其中特异性转移的材料是紫外线交联的。这些多肽中最大的与核仁核糖核酸内切酶的估计大小相对应，而分子量较小的物种可能代表部分蛋白水解酶。总的来说，这些结果表明核核核糖核酸内切酶的独特特异性可能部分归因于小鼠核糖体前RNA +650加工位点形成的稳定复合物，并且这种独特稳定复合物的形成提供了一种方法来特异性标记有限数量的可用部分纯化酶用于序列分析。
A nucleolar endoribonuclease from mouse Ehrlich ascites tumor cells, that has been implicated in the endonucleolytic cleavage of mouse precursor ribosomal RNA, specifically and stably binds an in vitroderived rRNA transcript containing the +650 early processing site. The specificity of binding was demonstrated by mobility shift analysis, glycerol gradient velocity sedimentation analysis, and UVcrosslinking studies. Binding did not require Mg2+and therefore was not dependent on cleavage; however, binding was dependent on the presence of the early +650 processing site since a pre-rRNA transcript with the +650 processing site deleted failed to compete in binding. A small nucleolar RNA component was not required for the formation of this stable complex or for the specific cleavage of a processing competent prerRNA transcript. UV crosslinking studies using 32Plabeled 5-azidouridine-substituted pre-rRNA with bound nucleolar endoribonuclease identified three closely sized polypeptides of ñ50, ñ48, and ñ45 kDa, respectively, that specifically crosslinked to the processing competent rRNA transcript. These three polypeptides species were identified following ribonuclease digestion and electrophoresis on a SDSpolyacrylamide gel. An identical pattern of labeled polypeptides was also identified from gel mobility shift analysis where the specifically shifted material was U.V. crosslinked. The largest of these polypeptides corresponded to the estimated size of the nucleolar endoribonuclease, while the lower molecular weight species may represent partially proteolyzed enzyme. Overall, these results suggest that the unique specificity of the nucleolar endoribonuclease may, in part, be attributed to the formation of a stable complex at the +650 processing site for mouse preribosomal RNA, and that formation of this unique stable complex affords a means to specifically label the limited amount of available partially purified enzyme for sequence analysis.