A 5' RNA stem-loop participates in the transcription attenuation mechanism that controls expression of the Bacillus subtilis trpEDCFBA operon.
A 5' RNA stem-loop participates in the transcription attenuation mechanism that controls expression of the Bacillus subtilis trpEDCFBA operon.
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5 RNA 茎环参与控制枯草芽孢杆菌 trpEDCFBA 操纵子表达的转录衰减机制。
DOI：
10.1128/jb.181.18.5742-5749.1999
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发表时间：
1999
影响因子：
3.2
通讯作者：
Babitzke,P
中科院分区：
文献类型：
--
作者：
Sudershana,S;Du,H;Mahalanabis,M;Babitzke,P
trpRNA结合衰减蛋白（TRAP）通过转录衰减调节枯草芽孢杆菌trpEDCFBA操纵子的表达。色氨酸激活的TRAP通过与11个（G/U）AG重复序列相互作用而与nascenttrpleader转录物结合。TRAP结合阻止了抗终止子结构的形成，从而促进了重叠终止子的形成，因此在RNA聚合酶到达trp结构基因之前终止了转录。除了抗终止子和终止子外，预期在trpleader转录物的5′端形成茎环结构。该结构的缺失导致atrpE′-′ lacZ翻译融合体表达的显著增加和响应色氨酸调节表达的能力降低。通过在5′茎环中引入一系列点突变，我们发现发夹的序列和结构对其调节功能都很重要，并且恢复碱基配对的补偿性变化部分恢复了野生型样表达水平。我们的研究结果表明，5′茎环功能主要是通过TRAP依赖的调节途径。凝胶位移结果表明，5 '茎环使TRAP对捕获RNA的亲和力增加了四到五倍，表明5'结构与TRAP相互作用。体外转录结果表明，这种5 '结构在减弱机制中发挥作用，因为茎环的缺失导致转录通读的增加。使用与5′茎环片段互补的寡核苷酸来证明5′结构的形成是该操纵子的适当减毒控制所必需的。
ThetrpRNA-binding attenuation protein (TRAP) regulates expression of theBacillus subtilis trpEDCFBAoperon by transcription attenuation. Tryptophan-activated TRAP binds to the nascenttrpleader transcript by interacting with 11 (G/U)AG repeats. TRAP binding prevents formation of an antiterminator structure, thereby promoting formation of an overlapping terminator, and hence transcription is terminated before RNA polymerase can reach thetrpstructural genes. In addition to the antiterminator and terminator, a stem-loop structure is predicted to form at the 5′ end of thetrpleader transcript. Deletion of this structure resulted in a dramatic increase in expression of atrpE′-′lacZtranslational fusion and a reduced ability to regulate expression in response to tryptophan. By introducing a series of point mutations in the 5′ stem-loop, we found that both the sequence and the structure of the hairpin are important for its regulatory function and that compensatory changes that restored base pairing partially restored wild-type-like expression levels. Our results indicate that the 5′ stem-loop functions primarily through the TRAP-dependent regulatory pathway. Gel shift results demonstrate that the 5′ stem-loop increases the affinity of TRAP fortrpleader RNA four- to fivefold, suggesting that the 5′ structure interacts with TRAP. In vitro transcription results indicate that this 5′ structure functions in the attenuation mechanism, since deletion of the stem-loop caused an increase in transcription readthrough. An oligonucleotide complementary to a segment of the 5′ stem-loop was used to demonstrate that formation of the 5′ structure is required for proper attenuation control of this operon.