2'-FLUORO-2'-DEOXYNUCLEOSIDE AND 2'-AMINO-2'-DEOXYNUCLEOSIDE 5'-TRIPHOSPHATES AS SUBSTRATES FOR T7 RNA-POLYMERASE
2'-FLUORO-2'-DEOXYNUCLEOSIDE AND 2'-AMINO-2'-DEOXYNUCLEOSIDE 5'-TRIPHOSPHATES AS SUBSTRATES FOR T7 RNA-POLYMERASE
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DOI：
10.1021/bi00155a016
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发表时间：
1992-10-13
期刊：
影响因子：
2.9
通讯作者：
ECKSTEIN, F
中科院分区：
文献类型：
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作者：
AURUP, H;WILLIAMS, DM;ECKSTEIN, F
2'-氟-和 2'-氨基-2'-脱氧核苷 5'-三磷酸已被研究作为 T7 RNA 聚合酶的底物。报告了 2'-氟-2'-脱氧尿苷、2'-氟-2'-脱氧胞苷和 2'-氨基-2'-脱氧尿苷掺入径流转录本的 Michaelis-Menten 动力学参数。尿苷的 2'-氨基衍生物是比 2'-氟衍生物更好的底物。凝胶电泳分析显示，虽然全长产物伴随有相当数量的较短片段，但用类似物可以获得长度为2500个核苷酸的全长转录物。根据动力学分析，2'-氨基尿苷三磷酸提供比 2'-氟类似物更清洁的产品。两个 tRNA 基因的转录表明，这种较短的模板可以转录为全长产物，基本上不会用任何类似物提前终止。
2'-Fluoro-and 2'-amino-2'-deoxynucleoside 5'-triphosphates have been investigated as substrates for T7 RNA polymerase. Michaelis-Menten kinetic parameters are reported for the incorporation of 2'-fluoro-2'-deoxyuridine, 2'-fluoro-2'-deoxycytidine, and 2'-amino-2'-deoxyuridine into runoff transcripts. The 2'-amino derivative of uridine is a better substrate than the 2'-fluoro derivative. Gel electrophoretic analysis shows that full-length transcripts with a length of 2500 nucleotides can be obtained with the analogues, although a considerable amount of shorter fragments accompanies the full-length product. In keeping with the kinetic analysis, the 2'-aminouridine triphosphate gives a cleaner product than the 2'-fluoro analogue. Transcription of two tRNA genes shows that such shorter templates can be transcribed to full-length products essentially without premature termination with any of the analogues.