Nuclear magnetic resonance studies of the hammerhead ribozyme domain. Secondary structure formation and magnesium ion dependence.
Nuclear magnetic resonance studies of the hammerhead ribozyme domain. Secondary structure formation and magnesium ion dependence.
复制标题
锤头核酶结构域的核磁共振研究。
DOI：
10.1016/0022-2836(91)90615-d
复制
发表时间：
1991
影响因子：
5.6
通讯作者：
Pardi,A
中科院分区：
文献类型：
--
作者：
Heus,HA;Pardi,A
质子核磁共振（n.m.r.）实验用于探测几种锤头RNA酶（核酶）结构域中的碱基对形成。锤头结构域由结合到互补的13个核苷酸的不可切割的DNA底物的34个核苷酸的核酶组成。三个锤头结构域进行了研究，不同的序列和稳定性的螺旋之一，参与识别底物的核酶。国家医学研究所数据显示核酶-底物复合物的化学计量为1：1。通过二维核Overhauser效应实验对锤头状配合物中的亚氨基质子共振进行了归属。这些数据证实了锤头结构域中三个螺旋区域中的两个的存在，从系统发育数据预测;并且也与第三螺旋的形成一致。由于锤头结构域的有效催化活性需要二价阳离子，因此n.m.r.研究了两种锤头状络合物的光谱。其中一种配合物在加入镁离子后显示出非常大的光谱变化。然而，在其中一个识别螺旋中具有最多C · G碱基对的复合物在加入镁后基本上没有光谱（因此推测结构）变化。这些数据与其中镁结合位点已经存在于无镁复合物中的模型一致，表明镁离子在本文所用的条件下主要起催化作用，而不是结构作用。
Proton nuclear magnetic resonance (n.m.r.) experiments were used to probe base-pair formation in several hammerhead RNA enzyme (ribozyme) domains. The hammerhead domains consist of a 34 nucleotide ribozyme bound to a complementary 13 nucleotide non-cleavable DNA substrate. Three hammerhead domains were studied that differ in the sequence and stability of one of the helices involved in recognition of the substrate by the ribozyme. The n.m.r. data show a 1 : 1 stoichiometry for the ribozyme-substrate complexes. The imino proton resonances in the hammerhead complexes were assigned by two-dimensional nuclear Overhauser effect experiments. These data confirm the presence of two of the three helical regions in the hammerhead domain, predicted from phylogenetic data; and are also consistent with the formation of the third helix. Since a divalent cation is required for efficient catalytic activity of the hammerhead domain, the magnesium ion dependence of the n.m.r. spectra was studied for two of the hammerhead complexes. One of the complexes showed very large spectral changes upon addition of magnesium ions. However, the complex that has the most C · G base-pairs in one of the recognition helices shows essentially no spectral (and therefore presumably structural) changes upon addition of magnesium. These data are consistent with a model where the magnesium binding site already exists in the magnesium-free complex, suggesting that the magnesium ion serves primarily a catalytic, and not a structural, role under the conditions used here.