In vitro and in vivo secondary structure probing of the thrS leader in Bacillus subtilis
In vitro and in vivo secondary structure probing of the thrS leader in Bacillus subtilis
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DOI：
10.1093/nar/26.23.5379
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发表时间：
1998-12-01
影响因子：
14.9
通讯作者：
Putzer, H
中科院分区：
文献类型：
--
作者：
Luo, D;Condon, C;Putzer, H
枯草芽孢杆菌thrS基因是革兰氏阳性菌中的T-box基因家族的成员，其表达受涉及直接tRNA：mRNA相互作用的tRNA介导的转录抗终止机制调节。这些基因的复杂前导序列只有很短的一级序列同源性，但通过比较这个家族的许多基因的前导序列，已经提出了一个共同的二级结构。tRNA：mRNA相互作用的机制在很大程度上依赖于二级结构模型，但到目前为止只得到遗传证据的支持。我们研究了B的结构。枯草杆菌thrS阅读器在溶液中，在保护实验中使用化学和酶的探针。还使用硫酸二甲酯在体内探测了thrS前导结构，并且体外和体内数据良好一致。我们已经将thrs前导序列组织成三个主要结构域，包括六个独立的茎环。在这个基因家族中，除了一个保守的短序列之外，所有的短序列都存在于环结构中。建议与tRNA（GGU）（Thr）isoacceptor相互作用的ACC指定密码子存在于凸起中，并且可能存在于堆叠构象中。所提出的抗终止子结构在含有终止子的转录物中不可见，但使用终止子的3 '-一半缺失的转录物进行探测，其折叠似乎与调控模型一致。该家族的其他基因的阅读序列，特别是特异性结构域，可以类似于实验解决的thrS结构进行折叠。
The Bacillus subtilis thrS gene is a member of the T-box gene family in Gram-positive organisms whose expression is regulated by a tRNA-mediated transcriptional antitermination mechanism involving a direct tRNA:mRNA interaction. The complex leader sequences of these genes share only short stretches of primary sequence homology, but a common secondary structure has been proposed by comparing the leaders of many genes of this family. The proposed mechanism for the tRNA:mRNA interaction depends heavily on the secondary structure model, but is so far only supported by genetic evidence. We have studied the structure of the B. subtilis thrS reader in solution, in protection experiments using both chemical and enzymatic probes. The thrS leader structure was also probed in vivo using dimethylsulphate and the in vitro and in vivo data are in good accordance. We have organized the thrS leader into three major domains comprising six separate stem-loops. All but one of the short sequences conserved in this gene family are present in loop structures. The ACC specifier codon proposed to interact with the tRNA(GGU)(Thr) isoacceptor is present in a bulge and probably exists in a stacking conformation. The proposed antiterminator structure is not visible in transcripts containing the terminator, but was probed using a transcript with the 3'-half of the terminator deleted and its folding appears consistent with the regulatory model. The reader sequences, and in particular the specifier domains, of the other genes of this family can be folded similarly to the experimentally solved thrS structure.