Ossenbühl F, Nickelsen J
Lehrstuhl für Allgemeine und Molekulare Botanik, Ruhr-Universität Bochum, 44780 Bochum, Germany.
Mol Cell Biol. 2000 Nov;20(21):8134-42. doi: 10.1128/MCB.20.21.8134-8142.2000.
Chloroplast translation is mediated by nucleus-encoded factors that interact with distinct cis-acting RNA elements. A U-rich sequence within the 5' untranslated region of the psbD mRNA has previously been shown to be required for its translation in Chlamydomonas reinhardtii. By using UV cross-linking assays, we have identified a 40-kDa RNA binding protein, which binds to the wild-type psbD leader, but is unable to recognize a nonfunctional leader mutant lacking the U-rich motif. RNA binding is restored in a chloroplast cis-acting suppressor. The functions of several site-directed psbD leader mutants were analyzed with transgenic C. reinhardtii chloroplasts and the in vitro RNA binding assay. A clear correlation between photosynthetic activity and the capability to bind RNA by the 40-kDa protein was observed. Furthermore, the data obtained suggest that the poly(U) region serves as a molecular spacer between two previously characterized cis-acting elements, which are involved in RNA stabilization and translation. RNA-protein complex formation depends on the nuclear Nac2 gene product that is part of a protein complex required for the stabilization of the psbD mRNA. The sedimentation properties of the 40-kDa RNA binding protein suggest that it interacts directly with this Nac2 complex and, as a result, links processes of chloroplast RNA metabolism and translation.
叶绿体翻译由与不同顺式作用RNA元件相互作用的核编码因子介导。先前已证明莱茵衣藻中psbD mRNA的5'非翻译区内富含U的序列是其翻译所必需的。通过紫外线交联试验，我们鉴定出一种40 kDa的RNA结合蛋白，它能与野生型psbD前导序列结合，但无法识别缺乏富含U基序的无功能前导序列突变体。在叶绿体顺式作用抑制子中RNA结合得以恢复。用转基因莱茵衣藻叶绿体和体外RNA结合试验分析了几个定点psbD前导序列突变体的功能。观察到光合活性与40 kDa蛋白结合RNA的能力之间存在明显的相关性。此外，所获得的数据表明，聚（U）区域作为两个先前已鉴定的顺式作用元件之间的分子间隔，这两个元件参与RNA稳定和翻译。RNA-蛋白质复合物的形成取决于核Nac2基因产物，该产物是psbD mRNA稳定所需的蛋白质复合物的一部分。40 kDa RNA结合蛋白的沉降特性表明它直接与该Nac2复合物相互作用，因此将叶绿体RNA代谢和翻译过程联系起来。
