trp RNA-binding attenuation protein-mediated long distance RNA refolding regulates translation of trpE in Bacillus subtilis
trp RNA-binding attenuation protein-mediated long distance RNA refolding regulates translation of trpE in Bacillus subtilis
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DOI：
10.1074/jbc.273.32.20494
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发表时间：
1998-08-07
影响因子：
4.8
通讯作者：
Babitzke, P
中科院分区：
文献类型：
--
作者：
Du, HS;Babitzke, P
trpEDCFBA 操纵子的表达在转录和翻译水平上受到枯草芽孢杆菌的 trp RNA 结合衰减蛋白 (TRAP) 的调节。当细胞含有足够水平的色氨酸来激活 TRAP 时，该蛋白在合成时与 trp 操纵子转录物结合，最常导致转录终止。然而，终止永远不是 100% 有效的，逃脱终止的转录本会受到翻译控制。我们确定 TRAP 介导的 trpE 翻译控制可以通过一种新型 RNA 构象转换机制发生。当 TRAP 与 trp 操纵子通读转录物的 5'-非翻译前导片段结合时，它可以破坏包含 TRAP 结合靶标部分的大型二级结构。这促进了 RNA 的重折叠，使得位于 TRAP 结合位点下游 100 多个核苷酸的 trpE Shine-Dalgarno 序列被隔离在稳定的 RNA 发夹中。无细胞翻译、核糖体趾印和 RNA 结构作图实验的结果表明，这种结构的形成通过阻止核糖体进入 trpE 核糖体结合位点来减少 TrpE 合成。通过检查多个核苷酸取代的效果（在不改变 Shine-Dalgarno 序列本身的情况下废除结构），证实了 Shine-Dalgarno 阻断发夹在控制 trpE 翻译中的作用。讨论了蛋白质介导的 RNA 重折叠作为控制基因表达的一般机制的可能性。
Expression of the trpEDCFBA operon is regulated at both the transcriptional and translational levels by the trp RNA-binding attenuation protein (TRAP) of Bacillus subtilis, When cells contain sufficient levels of tryptophan to activate TRAP, the protein binds to trp operon transcripts as they are being synthesized, most often causing transcription termination. However, termination is never 100% efficient, and transcripts that escape termination are subject to translational control. We determined that TRAP-mediated translational control of trpE can occur via a novel RNA conformational switch mechanism. When TRAP binds to the 5'-untranslated leader segment of a trp operon read-through transcript, it can disrupt a large secondary structure containing a portion of the TRAP binding target. This promotes refolding of the RNA such that the trpE Shine-Dalgarno sequence, located more than 100 nucleotides downstream from the TRAP binding site, becomes sequestered in a stable RNA hairpin. Results from cell-free translation, ribosome toeprint, and RNA structure mapping experiments demonstrate that formation of this structure reduces TrpE synthesis by blocking ribosome access to the trpE ribosome binding site. The role of the Shine-Dalgarno blocking hairpin in controlling translation of trpE was confirmed by examining the effect of multiple nucleotide substitutions that abolish the structure without altering the Shine-Dalgarno sequence itself. The possibility of protein-mediated RNA refolding as a general mechanism in controlling gene expression is discussed.