PROTON GRADIENT REGULATION 3 recognizes multiple targets with limited similarity and mediates translation and RNA stabilization in plastids
PROTON GRADIENT REGULATION 3 recognizes multiple targets with limited similarity and mediates translation and RNA stabilization in plastids
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DOI：
10.1111/j.1365-313x.2011.04593.x
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发表时间：
2011-07-01
期刊：
影响因子：
7.2
通讯作者：
Shikanai, Toshiharu
中科院分区：
文献类型：
--
作者：
Cai, Wenhe;Okuda, Kenji;Shikanai, Toshiharu
质子梯度调节 3 (PGR3) 包含 27 个五肽重复 (PPR) 基序，属于 P 亚家族。先前的研究表明，PGR3 在 petL 操纵子 RNA 的稳定化以及 petL 和编码叶绿体 NADH 脱氢酶样复合物 (NDH) 亚基的 11 个质体 ndh 基因中的一个或部分基因的翻译中发挥作用。 pgr3-3 等位基因被认为在假定的 PGR3 翻译功能中存在特别缺陷。在此，我们表明单顺反子 petL 转录物的多核糖体关联在 pgr3-3 中受损。我们检测到 petL 和 ndhA 的 5' 非翻译区 (UTR) 中弱保守的序列，并且这些假定的元件在体外被重组 PGR3 识别。此前，pgr3-2 被证明在稳定 petL 操纵子 RNA 和以野生型水平积累 NDH 方面存在特异性缺陷。与该 pgr3-2 表型一致，我们在此表明，第 12 个 PPR 基序中携带 pgr3-2 突变的重组蛋白与 ndhA 的 5' UTR 结合，但不与 petL 的 5' UTR 结合。这表明单个氨基酸的改变改变了 PGR3 的结合特异性。相比之下，在最后的第 27 个不完整 PPR 基序中携带 pgr3-3 突变的重组蛋白可以结合 petL 和 ndhA 5' UTR，这表明 PGR3 的 C 末端不是与靶标结合所必需的，但对于 petL 以及可能还有 ndhA 的翻译是必需的。我们的结果完全支持 PGR3 识别两个靶序列并参与多种功能的模型，即稳定 RNA 和激活翻译。
PROTON GRADIENT REGULATION 3 (PGR3) contains 27 pentatricopeptide repeat (PPR) motifs and belongs to the P-subfamily. Previous studies have suggested that PGR3 functions in the stabilization of petL operon RNA and also in the translation of petL and one, or some, of the 11 plastid ndh genes encoding subunits of chloroplast NADH dehydrogenase-like complex (NDH). The pgr3-3 allele has been suggested to be specifically defective in the putative PGR3 function of translation. Herein, we show that the polysome association of the monocistronic petL transcript is impaired in pgr3-3. We detected sequences weakly conserved in the 5' untranslated regions (UTRs) of petL and ndhA, and these putative elements were recognized by recombinant PGR3 in vitro. Previously, pgr3-2 was shown to be specifically defective in stabilizing petL operon RNA and to accumulate NDH at wild-type levels. Consistent with this pgr3-2 phenotype, we show here that a recombinant protein carrying the pgr3-2 mutation in the 12th PPR motif bound to the 5' UTR of ndhA but not of petL. This indicates that a single amino acid alteration changes the binding specificity of PGR3. In contrast, the recombinant protein carrying the pgr3-3 mutation in the final, 27th, incomplete PPR motif can bind to both petL and ndhA 5' UTRs, suggesting that the C-terminal end of PGR3 is not required for binding to targets but is essential for translation of petL and probably also ndhA. Our results fully support the model in which PGR3 recognizes two target sequences and is involved in multiple functions, i.e. stabilizing RNA and activating translation.