Kanai Akio, Sato Asako, Imoto Jun, Tomita Masaru
Institute for Advanced Biosciences, Keio University, Tsuruoka, 997-0017, Japan.
Biochem J. 2006 Jan 1;393(Pt 1):373-9. doi: 10.1042/BJ20050608.
Using a stem-loop RNA oligonucleotide (19-mer) containing an AUG sequence in the loop region as a probe, we screened the protein library from a hyperthermophilic archaeon, Pyrococcus furiosus, and found that a flavin-dependent thymidylate synthase, Pf-Thy1 (Pyrococcus furiosus thymidylate synthase 1), possessed RNA-binding activity. Recombinant Pf-Thy1 was able to bind to the stem-loop structure at a high temperature (75 degrees C) with an apparent dissociation constant of 0.6 microM. A similar stem-loop RNA structure was located around the translation start AUG codon of Pf-Thy1 RNA, and gel-shift analysis revealed that Pf-Thy1 could also bind to this stem-loop structure. In vitro translation analysis using chimaeric constructs containing the stem-loop sequence in their Pf-Thy1 RNA and a luciferase reporter gene indicated that the stem-loop structure acted as an inhibitory regulator of translation by preventing the binding of its Shine-Dalgarno-like sequence by positioning it in the stem region. Addition of Pf-Thy1 into the in vitro translation system also inhibited translation. These results suggested that this class of thymidylate synthases may autoregulate their own translation in a manner analogous to that of the well characterized thymidylate synthase A proteins, although there is no significant amino acid sequence similarity between them.
我们使用一种在环区含有AUG序列的茎环RNA寡核苷酸（19聚体）作为探针，从嗜热古菌激烈热球菌（Pyrococcus furiosus）中筛选蛋白质文库，发现一种黄素依赖性胸苷酸合酶Pf-Thy1（激烈热球菌胸苷酸合酶1）具有RNA结合活性。重组Pf-Thy1能够在高温（75℃）下与茎环结构结合，表观解离常数为0.6微摩尔。类似的茎环RNA结构位于Pf-Thy1 RNA的翻译起始AUG密码子周围，凝胶迁移分析表明Pf-Thy1也能与这种茎环结构结合。使用在其Pf-Thy1 RNA中含有茎环序列和荧光素酶报告基因的嵌合构建体进行的体外翻译分析表明，茎环结构通过将其类似Shine-Dalgarno序列定位在茎区域来阻止其结合，从而作为翻译的抑制调节因子。将Pf-Thy1添加到体外翻译系统中也会抑制翻译。这些结果表明，这类胸苷酸合酶可能以类似于已充分表征的胸苷酸合酶A蛋白的方式自动调节自身翻译，尽管它们之间没有明显的氨基酸序列相似性。
