Amiri K M, Hagerman P J
Department of Biochemistry, Biophysics and Genetics University of Colorado Health Science Center, Denver 80262, USA.
J Mol Biol. 1996 Aug 16;261(2):125-34. doi: 10.1006/jmbi.1996.0446.
The RNA "hammerhead" domain is a small element of secondary structure found in the genomes of certain plant pathogens. It possesses a core of conserved sequence at the conjunction of three helix stems, and is capable of undergoing self-cleavage in the presence of divalent cations. Both crystallographic and solution studies suggest that the domain is highly structured, with the three stems assuming a Y-shaped global conformation; however, such studies have employed either RNA analogues that were catalytically inactive, or conditions of temperature and pH for which rates of self-cleavage are slow. Thus, it was unknown whether such species represented the principal conformers during the cleavage process itself. In order to address this issue, a series of time-resolved, transient electric birefringence measurements was conducted in an effort to define the global conformation of an RNA hammerhead in real time throughout the process of self-cleavage. The current study demonstrates that the angular relationship between the two helices that flank the cleavage center is essentially unchanged between the pre-cleavage and post-cleavage forms. Moreover, despite the fact that at least one kinetic intermediate is formed during the self-cleavage reaction, there is no evidence for the existence of a significant population of intermediates with altered global conformation during cleavage. Thus, any conformational isomerism that may occur is likely to be relatively localized to the active center. Finally, it was observed that sequence elements lying outside of the conserved region, at the base of stem I, influence interhelix geometry. The current results are consistent with a structural model in which the active center possesses similar conformations pre-cleavage and post-cleavage. Such a model would help to explain the significant rate of reversal of the cleavage reaction (self-ligation).
RNA“锤头”结构域是在某些植物病原体基因组中发现的一种小的二级结构元件。它在三个螺旋茎的连接处具有保守序列核心，并且在二价阳离子存在下能够进行自我切割。晶体学和溶液研究均表明，该结构域具有高度结构化，三个茎呈现出Y形的整体构象；然而，此类研究要么使用了无催化活性的RNA类似物，要么采用了自我切割速率缓慢的温度和pH条件。因此，尚不清楚此类物种是否代表切割过程本身中的主要构象体。为了解决这个问题，进行了一系列时间分辨的瞬态电双折射测量，以实时确定RNA锤头在自我切割过程中的整体构象。当前研究表明，在切割前和切割后形式之间，位于切割中心两侧的两个螺旋之间的角度关系基本不变。此外，尽管在自我切割反应过程中至少形成了一种动力学中间体，但没有证据表明在切割过程中存在大量具有改变的整体构象的中间体。因此，任何可能发生的构象异构现象可能相对局限于活性中心。最后，观察到位于茎I底部保守区域之外的序列元件会影响螺旋间几何形状。当前结果与一种结构模型一致，即活性中心在切割前和切割后具有相似的构象。这样的模型将有助于解释切割反应（自我连接）的显著逆转速率。
