Padilla R, Sousa R
Department of Biochemistry, University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio,TX 78284-7760, USA.
Nucleic Acids Res. 1999 Mar 15;27(6):1561-3. doi: 10.1093/nar/27.6.1561.
A T7 RNAP mutant (Y639F) which eliminates discrimination of the chemical character of the NTP ribose 2'-group, facilitates incorporation of non-canonicalsubstrates into nucleic acids. However, transcripts containing a high percentage of non-canonical NMPs are poorly extended due to effects of the 2'-substituents on the transcript:template hybrid conformation. We tested the addition of compounds that stabilize A-type helix geometry to the reaction. High concentrations of polyamines, together with other changes in reaction conditions, greatly increased the synthesis of transcripts heavily substituted with non-canonical ribose 2'-groups. Template structures that facilitate promoter opening increased the efficiency of reactions where non-canonical substrates were incorporated during transcription of +1 to +6.
一种T7 RNA聚合酶突变体（Y639F）消除了对NTP核糖2'-基团化学性质的辨别，促进了非规范底物掺入核酸中。然而，由于2'-取代基对转录本：模板杂交构象的影响，含有高比例非规范NMP的转录本延伸较差。我们测试了向反应中添加稳定A型螺旋几何结构的化合物。高浓度的多胺以及反应条件的其他变化，极大地增加了被非规范核糖2'-基团大量取代的转录本的合成。促进启动子开放的模板结构提高了在转录+1至+6过程中掺入非规范底物的反应效率。
