De la Peña Marcos, Gago Selma, Flores Ricardo
Instituto de Biología Molecular y Celular de Plantas (UPV-CSIC), Universidad Politécnica de Valencia, Avenida de los Naranjos s/n, Valencia 46022, Spain.
EMBO J. 2003 Oct 15;22(20):5561-70. doi: 10.1093/emboj/cdg530.
Natural hammerhead ribozymes are mostly found in some viroid and viroid-like RNAs and catalyze their cis cleavage during replication. Hammerheads have been manipulated to act in trans and assumed to have a similar catalytic behavior in this artificial context. However, we show here that two natural cis-acting hammerheads self-cleave much faster than trans-acting derivatives and other reported artificial hammerheads. Moreover, modifications of the peripheral loops 1 and 2 of one of these natural hammerheads induced a >100-fold reduction of the self-cleavage constant, whereas engineering a trans-acting artificial hammerhead into a cis derivative by introducing a loop 1 had no effect. These data show that regions external to the central conserved core of natural hammerheads play a role in catalysis, and suggest the existence of tertiary interactions between these peripheral regions. The interactions, determined by the sequence and size of loops 1 and 2 and most likely of helices I and II, must result from natural selection and should be studied in order to better understand the hammerhead requirements in vivo.
天然锤头状核酶大多存在于一些类病毒和类病毒样RNA中，并在复制过程中催化其顺式切割。锤头状核酶已被改造用于反式作用，并假定在这种人工环境中具有类似的催化行为。然而，我们在此表明，两种天然顺式作用的锤头状核酶自我切割的速度比反式作用衍生物和其他已报道的人工锤头状核酶快得多。此外，对其中一种天然锤头状核酶的外周环1和环2进行修饰，导致自我切割常数降低了100倍以上，而通过引入环1将反式作用的人工锤头状核酶改造为顺式衍生物则没有效果。这些数据表明，天然锤头状核酶中央保守核心外部的区域在催化中起作用，并表明这些外周区域之间存在三级相互作用。由环1和环2以及很可能由螺旋I和螺旋II的序列和大小所决定的相互作用，必定是自然选择的结果，应该对其进行研究以便更好地理解锤头状核酶在体内的需求。
