Kopper R A, Stallcup T, Hufford G, Liarakos C D
Department of Chemistry, Hendrix College, Conway, AR 72032.
Nucleic Acids Res. 1994 Oct 25;22(21):4504-9. doi: 10.1093/nar/22.21.4504.
We used the chemical reagents dimethylsulfate and 4'-aminomethyl-4,5',8-trimethylpsoralen and the enzyme T1 ribonuclease to compare the 5'-end structure of ovalbumin mRNA in situ in purified hen oviduct nuclei and polysomes with that of the isolated mRNA. The qualitative pattern of structure-dependent base modifications and T1 ribonuclease cleavage sites in intranuclear and polysomal ovalbumin mRNAs was found to be nearly identical to those in isolated ovalbumin mRNA. These structural data are consistent with the presence of a trigonal stem-loop structure at the 5'-end of ovalbumin mRNA (hairpin-1) in nuclei and polysomes. Similar results were obtained for a coding region structure (hairpin-3) in intranuclear ovalbumin mRNA. We have recently shown that hairpin-1 positively affects the rate of ovalbumin mRNA translation in vitro and is part of a high affinity binding site for eucaryotic initiation factor-2 (eIF-2). The presence of hairpin-1 in ovalbumin mRNA in both a pretranslation state (nuclei) and active translation state (polysomes) is consistent with its hypothesized biological function as an intracellular initiation signal that facilitates the translation of this mRNA.
我们使用化学试剂硫酸二甲酯和4'-氨甲基-4,5',8-三甲基补骨脂素以及T1核糖核酸酶，来比较纯化的母鸡输卵管细胞核和多核糖体中卵清蛋白mRNA原位的5'-末端结构与分离出的mRNA的5'-末端结构。结果发现，核内和多核糖体中的卵清蛋白mRNA的结构依赖性碱基修饰和T1核糖核酸酶切割位点的定性模式，与分离出的卵清蛋白mRNA中的几乎相同。这些结构数据与核和多核糖体中卵清蛋白mRNA的5'-末端存在三角茎环结构（发夹-1）相一致。对于核内卵清蛋白mRNA中的编码区结构（发夹-3）也获得了类似结果。我们最近表明，发夹-1在体外对卵清蛋白mRNA的翻译速率有正向影响，并且是真核起始因子-2（eIF-2）高亲和力结合位点的一部分。卵清蛋白mRNA在翻译前状态（细胞核）和活跃翻译状态（多核糖体）中均存在发夹-1，这与其作为促进该mRNA翻译的细胞内起始信号的假定生物学功能相一致。
