Functional analysis of internal transcribed spacer 2 of Saccharomyces cerevisiae ribosomal DNA.
Functional analysis of internal transcribed spacer 2 of Saccharomyces cerevisiae ribosomal DNA.
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DOI：
10.1016/0022-2836(92)90251-e
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发表时间：
1992-02
影响因子：
5.6
通讯作者：
C. A. van der Sande;M. Kwa;R. V. van Nues;H. van Heerikhuizen;H. A. Raué;R. Planta
中科院分区：
文献类型：
--
作者：
C. A. van der Sande;M. Kwa;R. V. van Nues;H. van Heerikhuizen;H. A. Raué;R. Planta
利用之前描述的“标记核糖体”（pORCS）系统对酵母rDNA进行体内突变分析，我们发现ITS2 5 '端部分的小缺失完全阻断了26 S rRNA在29 S前体（5.8 S rRNA-ITS2 - 26 S rRNA）水平上的成熟。3 '端部分的各种缺失虽然严重降低了加工效率，但仍能形成一些成熟的26s rRNA。另一方面，ITS2缺失不影响成熟17s rRNA的产生。由于所有的缺失都严重干扰了最近提出的ITS2二级结构，这些发现表明ITS2的高阶结构在加工中起重要作用。全面或部分更换s的效果分析。cerevisiaeITS2与来自saccharomyces roseiorHansenula wingei的对应序列，指出二级结构模型的螺旋V是正确和有效处理的重要元素。直接突变分析表明，螺旋V中间碱基配对的中断对26s rRNA的形成没有明显影响。相反，在螺旋V的顶端引入聚类点突变，既破坏碱基配对，又改变环的序列，严重减少了加工。由于在环序列中只包含点突变的突变体产生正常数量的成熟26s rRNA，我们得出结论，螺旋V下端的精确（次级和/或初级）结构，但不包括环，对于有效去除ITS2至关重要。
Using the previously described “tagged ribosome” (pORCS) system forin vivomutational analysis of yeast rDNA, we show that small deletions in the 5′-terminal portion of ITS2 completely block maturation of 26 S rRNA at the level of the 29 Sbprecursor (5.8 S rRNA-ITS2–26 S rRNA). Various deletions in the 3′-terminal part, although severely reducing the efficiency of processing, still allow some mature 26 S rRNA to be formed. On the other hand, none of the ITS2 deletions affect the production of mature 17 S rRNA. Since all of the deletions severely disturb the recently proposed secondary structure of ITS2, these findings suggest an important role for higher order structure of ITS2 in processing.Analysis of the effect of complete or partial replacement ofS. cerevisiaeITS2 with its counterpart sequences fromSaccharomyces roseiorHansenula wingei, points to helix V of the secondary structure model as an important element for correct and efficient processing. Direct mutational analysis shows that disruption of base-pairing in the middle of helix V does not detectably affect 26 S rRNA formation. In contrast, introduction of clustered point mutations at the apical end of helix V that both disrupt base-pairing and change the sequence of the loop, severely reduces processing. Since a mutant containing only point mutations in the sequence of the loop produces normal amounts of mature 26 S rRNA, we conclude that the precise (secondary and/or primary) struture at the lower end of helix V, but excluding the loop, is of crucial importance for efficient removal of ITS2.