通过紫外光谱和圆二色光谱研究的自我剪接I组内含子中三螺旋结构域的合成模型。

Sarkar M, Sigurdsson S, Tomac S, Sen S, Rozners E, Sjöberg B M, Strömberg R, Gräslund A

Department of Biophysics, Arrhenius Laboratories, Stockholm University, Sweden.

Biochemistry. 1996 Apr 16;35(15):4678-88. doi: 10.1021/bi9523466.

Structural studies were performed on synthetic oligonucleotides with sequences corresponding to the P4/P6 and J3/4, J6/7 regions of the self-splicing group I intron of the bacteriophage T4 nrdB pre-mRNA, which correspond to the proposed triple-helical domain in the Tetrahymena thermophila intron. A 23-mer RNA was synthesized as a mixed ribo-deoxyribo oligonucleotide, modeling an expected base-paired region of P4 along with the J3/4 and P6 (5'-end bases of P6) regions. strand modeling the 3'-end bases of P6 and J6/7 regions, with which a triple helix may form, was synthesized as a pure oligoribonucleotide (7-mer RNA). The interactions of these oligonucleotides have been characterized by UV and circular dichroism (CD) spectroscopy. The results show that the 23-mer RNA forms a stable hairpin modeling the P4 base-paired region. Triple helix association between the 23-mer RNA hairpin and the 7-mer RNA single strand was detected by CD in the presence of Mg2+ (>5mM) but not in presence of a monovalent cation like Na+ (up to 500 mM). Studies on selected variants of both 7-mer and 23-mer RNAs were carried out. The results show that for the association of the two partner strands not only the formation of P6 helix but also triplet interactions between two strands are required. The association of the two strands in general follow a pattern predicted by comparative sequence analysis. Parallel studies on pure oligoribonucleotides having base sequence corresponding to those of the oligoribonucleotides showed no evidence of association under similar conditions, which could indicate that the 2'-hydroxyl groups of the riboses might play an important role in hydrogen bonding to form the required nucleoside triples. Molecular modeling studies on the proposed "plaited triple helix" formed by the association of the 23-mer RNA hairpin and 7-mer RNA single strand showed that the structure is sterically and energetically feasible.

对合成的寡核苷酸进行了结构研究，这些寡核苷酸的序列对应于噬菌体T4 nrdB前体mRNA的自我剪接I组内含子的P4/P6和J3/4、J6/7区域，它们对应于嗜热四膜虫内含子中提议的三螺旋结构域。合成了一种23聚体RNA，作为混合的核糖-脱氧核糖寡核苷酸，模拟P4的预期碱基配对区域以及J3/4和P6（P6的5'端碱基）区域。模拟P6和J6/7区域3'端碱基的链，可能与之形成三螺旋，被合成为纯寡核糖核苷酸（7聚体RNA）。这些寡核苷酸的相互作用已通过紫外和圆二色性（CD）光谱进行了表征。结果表明，23聚体RNA形成了一个稳定的发夹结构，模拟了P4碱基配对区域。在存在Mg2+（>5mM）时通过CD检测到23聚体RNA发夹与7聚体RNA单链之间的三螺旋缔合，但在存在单价阳离子如Na+（高达500 mM）时未检测到。对7聚体和23聚体RNA的选定变体进行了研究。结果表明，对于两条互补链的缔合，不仅需要形成P6螺旋，还需要两条链之间的三联体相互作用。两条链的缔合通常遵循比较序列分析预测的模式。对具有与寡核糖核苷酸碱基序列相对应的纯寡核糖核苷酸进行的平行研究表明，在类似条件下没有缔合的证据，这可能表明核糖的2'-羟基在形成所需的核苷三联体的氢键中可能起重要作用。对由23聚体RNA发夹和7聚体RNA单链缔合形成的提议的“编织三螺旋”进行的分子建模研究表明，该结构在空间和能量上是可行的。