Interhelix geometry of stems I and II of a self-cleaving hammerhead RNA.
Interhelix geometry of stems I and II of a self-cleaving hammerhead RNA.
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自裂解锤头 RNA 茎 I 和 II 的螺旋间几何形状。
DOI：
10.1021/bi00173a023
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发表时间：
1994
期刊：
影响因子：
2.9
通讯作者：
Hagerman,PJ
中科院分区：
文献类型：
--
作者：
Gast,FU;Amiri,KM;Hagerman,PJ
为了研究自切割锤头结构域的几何形状，已经构建了RNA异源双链体，其中结构域的三个螺旋茎中的两个各自被延长至76个双链体碱基对（bp），导致约100 bp的RNA分子。160 bp。异源双链分子能够在中性pH下，在添加Mg 2+或Mn 2+时进行自切割，但在切割后不解离。使用电泳和流体动力学方法的组合，如先前用于定义四向DNA分支的几何形状[Copper & Hagerman（1989）Proc.Natl. Acad. Sci. USA 86，7336-7340]，我们已经确定细长锤头柄在自劈裂之前几乎共线。自裂解后，在不存在Mg 2+的情况下，两个茎之间的角度变得更加尖锐和/或灵活;然而，在Mg 2+的存在下，裂解结构似乎保留了预裂解形式的几何形状（至少相对于茎间角）。还观察到Mn 2+比Mg 2+更大程度地促进自裂解反应，这与早期的观察结果一致。最后，虽然在未切割的分子中，伸长的螺旋似乎几乎是共线的，但相对于线性对照RNA，该物种的电泳迁移率显著降低，这表明仅从凝胶阻滞实验中注意到这一点。
In order to investigate the geometry of a self-cleaving hammerhead domain, an RNA heteroduplex has been constructed in which two of the three helix stems of the domain have each been elongated to 76 duplex base pairs (bp), resulting in an RNA molecule of ca. 160 bp. The heteroduplex molecule is capable of undergoing self-cleavage at neutral pH, upon addition of either Mg2+ or Mn2+, but does not dissociate following cleavage. Using a combination of electrophoretic and hydrodynamic methods, as employed earlier to define the geometry of a four-way DNA branch [Copper & Hagerman (1989) Proc. Natl. Acad. Sci. USA 86, 7336-7340], we have determined that the elongated hammerhead stems are nearly collinear prior to self-cleavage. Following self-cleavage, and in the absence of Mg2+, the angle between the two stems becomes much more acute and/or flexible; however, in the presence of Mg2+, the cleaved structure appears to retain the geometry of the precleaved form (at least with respect to the interstem angle). It is also observed that the self-cleavage reaction is promoted by Mn2+ to a much greater extent than by Mg2+, consistent with earlier observations. Finally, although the elongated helices appear to be nearly collinear in the uncleaved molecule, the electrophoretic mobility of that species is dramatically reduced with respect to linear control RNAs, indicating thatcaution si angles solely from gel retardation experiments.