The chemical basis of adenosine conservation throughout the Tetrahymena ribozyme
The chemical basis of adenosine conservation throughout the Tetrahymena ribozyme
复制标题
DOI：
10.1017/s1355838298980086
复制
发表时间：
1998-05-01
期刊：
影响因子：
4.5
通讯作者：
Strobel, SA
中科院分区：
文献类型：
--
作者：
Ortoleva-Donnelly, L;Szewczak, AA;Strobel, SA
腺苷在几个RNA结构基序中以不成比例的高频率存在，为了探索单个腺苷功能基团对I组内含子活性的重要性，我们对一系列腺苷类似物进行了核苷酸模拟干扰图谱(NAIM)。本文报道了用于NAIM的8个标记有α-硫代硫酸酯的腺苷衍生物的合成、转录掺入和在四膜虫I组内含子中观察到的干扰模式，所有类似物作为A准确地结合到转录本中，干扰四膜虫内含子3‘-外显子连接反应的位置与系统发育保守的位置重合，然而每个内含子的干扰模式是不同的，这些干扰数据提供了几个生化约束条件，改善了我们对四膜虫核酶结构的理解，例如，这些数据支持J6/6a区域内的一个必要的A平台，P3和P7螺旋的主槽填充，P3和J4/5螺旋的小槽填充，以及鸟苷辅因子结合的轴向模型。这些数据还识别了内含子核心(J8/7)核心高度保守的单链区域内的几个基本官能团，在内含子的四个位置，2‘-氟A可以观察到干扰，但不能与2’-脱氧A，2‘-脱氧A，根据与P4-P6晶体结构的比较，这可能为核糖采用必要的C2‘-Endo构象的核苷酸位置提供生化特征。在与2‘-脱氧A有干扰的其他情况下，2’-氟A干扰的存在或不存在有助于确定2‘-OH是作为氢键供体还是受体，四膜虫内含子的作图建立了一组信息的基础，这将使这些试剂能够在不太了解的系统中放心地使用。
Adenosines are present at a disproportionately high frequency within several RNA structural motifs, To explore the importance of individual adenosine functional groups for group I intron activity, we performed Nucleotide Analog Interference Mapping (NAIM) with a collection of adenosine analogues, This paper reports the synthesis, transcriptional incorporation, and the observed interference pattern throughout the Tetrahymena group I intron for eight adenosine derivatives tagged with an alpha-phosphorothioate linkage for use in NAIM, All of the analogues were accurately incorporated into the transcript as an A, The sites that interfere with the 3'-exon ligation reaction of the Tetrahymena intron are coincident with the sites of phylogenetic conservation, yet the interference patterns for each analogue are different, These interference data provide several biochemical constraints that improve our understanding of the Tetrahymena ribozyme structure, For example, the data support an essential A-platform within the J6/6a region, major groove packing of the P3 and P7 helices, minor groove packing of the P3 and J4/5 helices, and an axial model for binding of the guanosine cofactor, The data also identify several essential functional groups within a highly conserved single-stranded region in the core of the intron (J8/7), At four sites in the intron, interference was observed with 2'-fluoro A, but not with 2'-deoxy A, Based upon comparison with the P4-P6 crystal structure, this may provide a biochemical signature for nucleotide positions where the ribose sugar adopts an essential C2'-endo conformation. In other cases where there is interference with 2'-deoxy A, the presence or absence of 2'-fluoro A interference helps to establish whether the 2'-OH acts as a hydrogen bond donor or acceptor, Mapping of the Tetrahymena intron establishes a basis set of information that will allow these reagents to be used with confidence in systems that are less well understood.