Maglott E J, Glick G D
Department of Chemistry, University of Michigan, Ann Arbor, MI 48109-1055, USA.
Nucleic Acids Res. 1998 Mar 1;26(5):1301-8. doi: 10.1093/nar/26.5.1301.
Three analogs of unmodified yeast tRNAPhe, each possessing a single disulfide cross-link, have been designed and synthesized. One cross-link is between G1 and C72 in the amino acid acceptor stem, a second cross-link is in the central D region of yeast tRNAPhe between C11 and C25 and the third cross-link bridges U16 and C60 at the D loop/T loop interface. Air oxidation to form the cross-links is quantitative and analysis of the cross-linked products by native and denaturing PAGE, RNase T1 mapping, Pb(II) cleavage, UV cross-linking and thermal denaturation demonstrates that the disulfide bridges do not alter folding of the modified tRNAs relative to the parent sequence. The finding that cross-link formation between thiol-derivatized residues correlates with the position of these groups in the crystal structure of native yeast tRNAPhe and that the modifications do not significantly perturb native structure suggests that this methodology should be applicable to the study of RNA structure, conformational dynamics and folding pathways.
已设计并合成了三种未修饰的酵母苯丙氨酸转运核糖核酸（tRNAPhe）类似物，每种都具有一个二硫键交联。一个交联位于氨基酸接受茎中的G1和C72之间，第二个交联位于酵母苯丙氨酸转运核糖核酸中央D区域的C11和C25之间，第三个交联在D环/T环界面处连接U16和C60。通过空气氧化形成交联是定量的，并且通过天然和变性聚丙烯酰胺凝胶电泳（PAGE）、核糖核酸酶T1图谱分析、Pb（II）切割、紫外线交联和热变性对交联产物进行分析表明，相对于亲本序列，二硫键桥不会改变修饰的转运核糖核酸的折叠。硫醇衍生化残基之间交联形成与这些基团在天然酵母苯丙氨酸转运核糖核酸晶体结构中的位置相关，并且修饰不会显著干扰天然结构，这一发现表明该方法应适用于RNA结构、构象动力学和折叠途径的研究。
