Hwang S P, Eisenberg M, Binder R, Shelness G S, Williams D L
Department of Pharmacological Sciences, State University of New York, Stony Brook 11794.
J Biol Chem. 1989 May 15;264(14):8410-8.
Analyses of apolipoprotein II mRNA with chemical and enzymatic probes showed that double- and single-stranded regions were distributed uniformly along the mRNA except for a large (72 nucleotides) single-stranded region containing the translation stop codon. Secondary structure models constrained by the experimental data were made by varying the distance (along the mRNA) over which base pairing was allowed. Four prominent secondary structures were seen with restrictions of 165, 330, or 659 nucleotides suggesting that such structures from via local interactions over distances of 50-120 nucleotides. Predicted long range interactions involve only 2-3 base pairs while local interactions involve helices of 4-10 base pairs. Predicted helices of greater than or equal to 4 base pairs occur primarily within exons, raising the possibility that prominent secondary structures in mRNAs may be largely due to intraexonic base pairing. Tests of single- and double-stranded domains by oligonucleotide-directed RNase H cleavage and primer extension were in accord with the structure model and with nuclease and chemical modification data. The model predicting base pairing between the coding and the 3' noncoding regions was tested by RNase H cleavage followed by oligo(dT)-cellulose chromatography to separate 5' and 3' mRNA fragments. Most (82%) of the 5' fragment remained associated with the 3' noncoding region in a structure with a tm = 50 degrees C in 0.2 M Na+ suggesting that this stem could be stable in vivo. This stem may be stable in the isolated mRNA, but would likely occur transiently in polyribosomal apolipoprotein II mRNA due to ribosome transit through the 5' side of the stem. Alternate structures may occur in this region during ribosome transit and play a role in translation termination or in determining the susceptibility of the mRNA to degradation.
用化学和酶促探针分析载脂蛋白II信使核糖核酸（mRNA）表明，除了包含翻译终止密码子的一个大的（72个核苷酸）单链区外，双链区和单链区沿mRNA均匀分布。通过改变允许碱基配对的距离（沿mRNA），构建了受实验数据约束的二级结构模型。当限制碱基配对距离为165、330或659个核苷酸时，可观察到四种显著的二级结构，这表明此类结构是通过50 - 120个核苷酸距离的局部相互作用形成的。预测的长程相互作用仅涉及2 - 3个碱基对，而局部相互作用涉及4 - 10个碱基对的螺旋。预测的大于或等于4个碱基对的螺旋主要出现在外显子内，这增加了mRNA中显著二级结构可能主要归因于外显子内碱基配对的可能性。通过寡核苷酸定向核糖核酸酶H切割和引物延伸对单链和双链结构域进行的测试与结构模型以及核酸酶和化学修饰数据一致。通过核糖核酸酶H切割，随后用寡聚（dT）-纤维素柱层析分离5'和3'mRNA片段，对预测的编码区和3'非编码区之间的碱基配对模型进行了测试。5'片段的大部分（82%）在0.2M Na+中tm = 50℃的结构中仍与3'非编码区结合，这表明该茎在体内可能是稳定的。该茎在分离的mRNA中可能是稳定的，但由于核糖体穿过茎的5'侧，在多核糖体载脂蛋白II mRNA中可能会短暂出现。在核糖体穿过过程中，该区域可能会出现交替结构，并在翻译终止或确定mRNA对降解的敏感性中发挥作用。
