The global conformation of the hammerhead ribozyme determined using residual dipolar couplings
The global conformation of the hammerhead ribozyme determined using residual dipolar couplings
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DOI：
10.1021/bi012167q
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发表时间：
2002-10-01
期刊：
影响因子：
2.9
通讯作者：
Pardi, A
中科院分区：
文献类型：
--
作者：
Bondensgaard, K;Mollova, ET;Pardi, A
锤头状核酶的全局结构的情况下，Mg 2+的溶液NMR实验确定。锤头状核酶基序形成由连接到催化核心的三个螺旋茎组成的分支结构。H-1-N-15和H-1-C-13残余偶极偶联在一组与未标记的不可裂解底物复合的差异N-15/C-13标记的核酶中测量。残余偶极耦合提供分子的局部和全局结构的取向信息。残余偶极耦合的分析表明，在溶液中的三个螺旋茎的局部结构是很好地模拟了A型构象。然而，在没有Mg 2+的情况下，锤头在溶液中的全局结构与锤头的晶体结构中观察到的Y形构象不一致。将螺旋茎的残余偶极耦合与来自催化核心的标准NOE和J耦合常数NMR数据相结合。NOE数据显示结构域2中剪切G-A碱基对的形成。这些NMR数据被用来确定锤头中三个螺旋柄的全局取向。锤头在这些条件下形成了一个相当延伸的结构，茎I和II之间的角度很大（近似153度），茎II和III之间的角度较小（近似100度），茎I和III之间的角度最小（近似77度）。残余偶极耦合数据还包含分子动力学的信息，并在这里使用锤头状核酶-底物复合物中的螺旋结构域的灵活性提供定性信息。
The global structure of the hammerhead ribozyme was determined in the absence of Mg2+ by solution NMR experiments. The hammerhead ribozyme motif forms a branched structure consisting of three helical stems connected to a catalytic core. The H-1-N-15 and H-1-C-13 residual dipolar couplings were measured in a set of differentially N-15/C-13-labeled ribozymes complexed with an unlabeled noncleavable substrate. The residual dipolar couplings provide orientation information on both the local and the global structure of the molecule. Analysis of the residual dipolar couplings demonstrated that the local structure of the three helical stems in solution is well modeled by an A-form conformation. However, the global structure of the hammerhead in solution in the absence of Mg2+ is not consistent with the Y-shaped conformation observed in crystal structures of the hammerhead. The residual dipolar couplings for the helical stems were combined with standard NOE and J coupling constant NMR data from the catalytic core. The NOE data show formation of sheared G-A base pairs in domain 2. These NMR data were used to determine the global orientation of the three helical stems in the hammerhead. The hammerhead forms a rather extended structure under these conditions with a large angle between stems I and II (similar to153degrees), a smaller angle between stems II and III (similar to100degrees), and the smallest angle between steins I and III (similar to77degrees). The residual dipolar coupling data also contain information on the dynamics of the molecule and were used here to provide qualitative information on the flexibility of the helical domains in the hammerhead ribozyme-substrate complex.