Fang Peng, Spevak Christina C, Wu Cheng, Sachs Matthew S
Department of Environmental and Biomolecular Systems, OGI School of Science & Engineering, Oregon Health & Science University, Beaverton, OR 97006-8921, USA.
Proc Natl Acad Sci U S A. 2004 Mar 23;101(12):4059-64. doi: 10.1073/pnas.0400554101. Epub 2004 Mar 12.
The evolutionarily conserved fungal arginine attenuator peptide (AAP), as a nascent peptide, stalls the translating ribosome in response to the presence of a high concentration of the amino acid arginine. Here we examine whether the AAP maintains regulatory function in fungal, plant, and animal cell-free translation systems when placed as a domain near the N terminus or internally within a large polypeptide. Pulse-chase analyses of the radiolabeled polypeptides synthesized in these systems indicated that wild-type AAP functions at either position to stall polypeptide synthesis in response to arginine. Toeprint analyses performed to map the positions of stalled ribosomes on transcripts introduced into the fungal system revealed that ribosome stalling required translation of the AAP coding sequence. The positions of the stalled ribosomes were consistent with the sizes of the radiolabeled polypeptide intermediates. These findings demonstrate that an internal polypeptide domain in a nascent chain can regulate eukaryotic translational elongation in response to a small molecule. Apparently the peptide-sensing features are conserved in fungal, plant, and animal ribosomes. These data provide precedents for translational strategies that would allow domains within nascent polypeptide chains to modulate gene expression.
进化上保守的真菌精氨酸衰减肽（AAP）作为一种新生肽，在高浓度氨基酸精氨酸存在时会使翻译中的核糖体停滞。在这里，我们研究当AAP作为一个结构域置于靠近N端或位于一个大的多肽内部时，它在真菌、植物和动物无细胞翻译系统中是否保持调节功能。对在这些系统中合成的放射性标记多肽进行脉冲追踪分析表明，野生型AAP在任一位置起作用，以响应精氨酸而使多肽合成停滞。为了绘制引入真菌系统的转录本上停滞核糖体的位置而进行的足迹分析表明，核糖体停滞需要AAP编码序列的翻译。停滞核糖体的位置与放射性标记多肽中间体的大小一致。这些发现表明，新生链中的内部多肽结构域可以响应小分子调节真核生物的翻译延伸。显然，肽感知特征在真菌、植物和动物核糖体中是保守的。这些数据为允许新生多肽链内的结构域调节基因表达的翻译策略提供了先例。
