Ito Yuko, Sone Yumiko, Mizutani Takaharu
Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya 467-8603, Japan.
Mol Biol Rep. 2004 Mar;31(1):31-6. doi: 10.1023/b:mole.0000013501.82852.40.
A non-Watson-Crick G-A/A-G base pair is found in SECIS (selenocysteine-insertion sequence) element in the 3'-untranslated region of Se-protein mRNAs and in the functional site of the hammerhead ribozyme. We studied the stability of G-A/A-G base pair (bold) in 17mer GT(U)GACGGAAACCGGAAC synthetic DNA and RNA oligonucleotides by thermal melting experiments and gel electrophoresis. The measured Tm value of DNA oligonucleotide having G-A/A-G pair showed an intermediate value (58 degrees C) between that of Watson-Crick G-C/C-G base pair (75 degrees C) and that of G-G/A-A of non-base-pair (40 degrees C). Similar thermal melting patterns were obtained with RNA oligonucleotides. This result indicates that the secondary structure of oligonucleotide having G-A/A-G base pair is looser than that of the G-C type Watson-Crick base pair. In the comparison between RNA and DNA having G-A/A-G base pair, the Tm value of the RNA oligonucleotide was 11 degrees C lower than that of DNA, indicating that DNA has a more rigid structure than RNA. The stained pattern of oligonucleotide on polyacrylamide gel clarified that the mobility of the DNA oligonucleotide G-A/A-G base pair changed according to the urea concentration from the rigid state (near the mobility of G-C/C-G oligonucleotide) in the absence of urea to the random state (near the mobility of G-G/A-A oligonucleotide) in 7 M urea. However, the RNA oligonucleotide with G-A/A-G pair moved at an intermediate mobility between that of oligonucleotide with G-C/C-G and of the oligonucleotide with G-G/A-A, and the mobility pattern did not depend on urea concentration. Thus, DNA and RNA oligonucleotides with the G-A/A-G base pair showed a pattern indicating an intermediate structure between the rigid Watson-Crick base pair and the random structure of non-base pair. RNA with G-A/A-G base pair has the intermediate structure not influenced by urea concentration. Finally, this study indicated that the intermediate rigidity imparted by Non-Watson-Crick base pair in SECIS element plays an important role in the selenocysteine expression by UGA codon.
在硒蛋白mRNA 3'非翻译区的硒代半胱氨酸插入序列（SECIS）元件以及锤头状核酶的功能位点中发现了非沃森-克里克G-A/A-G碱基对。我们通过热变性实验和凝胶电泳研究了17聚体GT(U)GACGGAAACCGGAAC合成DNA和RNA寡核苷酸中G-A/A-G碱基对（加粗）的稳定性。含有G-A/A-G对的DNA寡核苷酸的测量熔解温度（Tm）值显示为中间值（58℃），介于沃森-克里克G-C/C-G碱基对（75℃）和非碱基对G-G/A-A（40℃）之间。RNA寡核苷酸也得到了类似的热变性模式。这一结果表明，含有G-A/A-G碱基对的寡核苷酸的二级结构比G-C型沃森-克里克碱基对的二级结构更松散。在比较含有G-A/A-G碱基对的RNA和DNA时，RNA寡核苷酸的Tm值比DNA低11℃，表明DNA的结构比RNA更刚性。聚丙烯酰胺凝胶上寡核苷酸的染色模式表明，DNA寡核苷酸G-A/A-G碱基对的迁移率根据尿素浓度而变化，从无尿素时的刚性状态（接近G-C/C-G寡核苷酸的迁移率）到7M尿素中的随机状态（接近G-G/A-A寡核苷酸的迁移率）。然而，含有G-A/A-G对的RNA寡核苷酸的迁移率介于含有G-C/C-G的寡核苷酸和含有G-G/A-A的寡核苷酸之间，且迁移率模式不依赖于尿素浓度。因此，含有G-A/A-G碱基对的DNA和RNA寡核苷酸呈现出一种介于刚性沃森-克里克碱基对和非碱基对随机结构之间的中间结构模式。含有G-A/A-G碱基对的RNA具有不受尿素浓度影响的中间结构。最后，这项研究表明，SECIS元件中非沃森-克里克碱基对赋予的中间刚性在UGA密码子介导的硒代半胱氨酸表达中起重要作用。
