Structure and function of the conserved 690 hairpin in Escherichia coli 16 S ribosomal RNA: analysis of the stem nucleotides.
Structure and function of the conserved 690 hairpin in Escherichia coli 16 S ribosomal RNA: analysis of the stem nucleotides.
复制标题
DOI：
10.1006/jmbi.2000.3852
复制
发表时间：
2000-06
影响因子：
5.6
通讯作者：
S. Morosyuk;K. Lee;J. SantaLucia;P. Cunningham
中科院分区：
文献类型：
--
作者：
S. Morosyuk;K. Lee;J. SantaLucia;P. Cunningham
大肠杆菌 16 S rRNA 的 680 至 710 号核苷酸形成核糖体功能所需的独特结构域。本研究的目的是确定 690 区域配对相互作用的功能意义。基于系统发育和化学修饰研究，为该发夹提出了两种不同的二级结构。为了研究 690 发夹中的配对相互作用对核糖体功能的影响，并确定哪些拟议的二级结构具有生物学意义，我们进行了即时进化实验，其中形成拟议的碱基对和 690 茎的悬挂端的九个核苷酸被随机突变，并选择了功能突变体组合。总共分离出了 96 个独特的功能突变体，并进行了体内分析和测序。对这些数据的分析揭示了突变核苷酸之间广泛的碱基配对和堆积相互作用。核糖体功能绝对需要在位置 688 和 699 之间形成 Watson-Crick 碱基对或 G.U 对。我们还对 31 核苷酸 RNA 进行了 NMR 研究，表明在 688 和 699 号核苷酸之间形成了功能上重要的碱基对。然而，在 689 和 698 号位置之间形成第二个碱基对对于核糖体功能来说并不是必需的，但核糖体功能水平与这些位置的核苷酸对的预测热力学稳定性相关。普遍保守的位置 G690 和 U697 通常被描述为形成 G.U 错配。我们的数据显示这些位置之间的共变，但不支持 G690:U697 对形成摆动结构的假设。 14-nt 和 31-nt RNA 模型的 NMR 研究支持了这些发现，并表明 G690 和 U697 参与了不寻常的堆积相互作用，但不形成摆动对。初步核磁共振结构分析表明，690 发夹的环部分折叠成高度结构化的新颖构象。
Nucleotides 680 to 710 of Escherichia coli 16 S rRNA form a distinct structural domain required for ribosome function. The goal of this study was to determine the functional significance of pairing interactions in the 690 region. Two different secondary structures were proposed for this hairpin, based on phylogenetic and chemical modification studies. To study the effect of pairing interactions in the 690 hairpin on ribosome function and to determine which of the proposed secondary structures is biologically significant, we performed an instant-evolution experiment in which the nine nucleotides that form the proposed base-pairs and dangling ends of the 690 stem were randomly mutated, and functional mutant combinations were selected. A total of 96 unique functional mutants were isolated, assayed in vivo, and sequenced. Analysis of these data revealed extensive base-pairing and stacking interactions among the mutated nucleotides. Formation of either a Watson-Crick base-pair or G.U pair between positions 688 and 699 is absolutely required for ribosome function. We also performed NMR studies of a 31-nucleotide RNA which indicate the formation of a functionally important base-pair between nucleotides 688 and 699. Formation of a second base-pair between positions 689 and 698, however, is not essential for ribosome function, but the level of ribosome function correlates with the predicted thermodynamic stability of the nucleotide pairs in these positions. The universally conserved positions G690 and U697 are generally portrayed as forming a G.U mismatch. Our data show co-variation between these positions, but do not support the hypothesis that the G690:U697 pair forms a wobble structure. NMR studies of model 14-nt and 31-nt RNAs support these findings and show that G690 and U697 are involved in unusual stacking interactions but do not form a wobble pair. Preliminary NMR structural analysis reveals that the loop portion of the 690 hairpin folds into a highly structured and novel conformation.