Nucleotide analog interference mapping of the hairpin ribozyme: implications for secondary and tertiary structure formation.
Nucleotide analog interference mapping of the hairpin ribozyme: implications for secondary and tertiary structure formation.
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发夹核酶的核苷酸类似物干扰图谱：对二级和三级结构形成的影响。
DOI：
10.1006/jmbi.1999.2959
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发表时间：
1999
影响因子：
5.6
通讯作者：
S. Strobel
中科院分区：
文献类型：
--
作者：
S. Ryder;S. Strobel
发夹核酶是一种天然存在的小RNA，能够折叠成独特的三维结构并催化特定的磷酸二酯转移反应。我们采用了一种高通量筛选程序，称为核苷酸类似物干扰映射（NAIM），以确定对该核酶的正确折叠和催化重要的功能基团。共使用18个磷酸硫酸标记的核苷酸类似物来确定单个核糖2'-OH和嘌呤官能基对发夹核酶连接反应的贡献。2'-脱氧核苷酸类似物的取代破坏了核酶内6个位点的活性，并且在11个保守的嘌呤核苷酸中每一个都观察到独特的干扰模式。在大多数可获得此类信息的情况下，NAIM数据与先前报道的单位点替代结果一致。通过与分离的A环和B环核磁共振结构和完整核酶模型的比较，解释了干扰模式。这些数据提供了生物化学证据，支持了每个环中大部分（但不是全部）非规范碱基对，并确定了最有可能参与环A和环b之间三级界面的官能团，这些官能团包括A10、G11、U12、C25和A38的2'-OH基团，G11的外环胺，以及A9和A24的小凹槽边缘。数据还预测了U39和U41的非a型糖袋几何形状。在此基础上，提出了环a与环B三级相互作用的修正模型。本工作明确了发夹核酶嘌呤核苷酸保守的化学基础，为干扰抑制实验的设计和解释提供了依据。
The hairpin ribozyme is a small, naturally occurring RNA capable of folding into a distinct three-dimensional structure and catalyzing a specific phosphodiester transfer reaction. We have adapted a high throughput screening procedure entitled nucleotide analog interference mapping (NAIM) to identify functional groups important for proper folding and catalysis of this ribozyme. A total of 18 phosphorothioate-tagged nucleotide analogs were used to determine the contribution made by individual ribose 2'-OH and purine functional groups to the hairpin ribozyme ligation reaction. Substitution with 2'-deoxy-nucleotide analogs disrupted activity at six sites within the ribozyme, and a unique interference pattern was observed at each of the 11 conserved purine nucleotides. In most cases where such information is available, the NAIM data agree with the previously reported single-site substitution results. The interference patterns are interpreted in comparison to the isolated loop A and loop B NMR structures and a model of the intact ribozyme. These data provide biochemical evidence in support of many, but not all, of the non-canonical base-pairs observed by NMR in each loop, and identify the functional groups most likely to participate in the tertiary interface between loop A and loop B. These groups include the 2'-OH groups of A10, G11, U12, C25, and A38, the exocyclic amine of G11, and the minor groove edge of A9 and A24. The data also predict non-A form sugar pucker geometry at U39 and U41. Based upon these results, a revised model for the loop A tertiary interaction with loop B is proposed. This work defines the chemical basis of purine nucleotide conservation in the hairpin ribozyme, and provides a basis for the design and interpretation of interference suppression experiments.
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影响因子：
5.6
作者：
Nesbitt, SM;Erlacher, HA;Fedor, MJ
通讯作者：
Fedor, MJ
通过核苷酸类似物干扰图谱定义四膜虫 I 组内含子功能所必需的化学基团。
DOI：
10.1073/pnas.94.7.2903
发表时间：
1997
影响因子：
11.1
作者：
Strobel,SA;Shetty,K
通讯作者：
Shetty,K
影响因子：
--
作者：
Scott A. Strobel;L. Ortoleva-Donnelly
通讯作者：
Scott A. Strobel;L. Ortoleva-Donnelly
影响因子：
11.4
作者：
SOUSA, R;PADILLA, R
通讯作者：
PADILLA, R
影响因子：
4.5
作者：
Ortoleva-Donnelly, L;Szewczak, AA;Strobel, SA
通讯作者：
Strobel, SA