Molecular analysis of a synthetic tetracycline-binding riboswitch
Molecular analysis of a synthetic tetracycline-binding riboswitch
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作者：
Hanson, S;Bauer, G;Suess, B
核糖开关是新近发现的调控元件，其仅由RNA组成，在预先形成的结合口袋中感测其配体，并响应于配体结合而执行构象转换，从而导致改变的基因表达。当四环素（tc）结合适体插入报告基因的5'非翻译区（UTR）时，其调节表现出核糖开关的所有特征。化学和酶的探测表明，适体由两个茎，P1和P2，这是已经存在的TC的情况下，形成的适体的支架。它们被凸起B1-2和相对的茎环（P3-L3）分开。探测模式中的Tc依赖性变化仅出现在凸起B1-2（核苷酸9-13）和环L3的上部。饱和诱变证实了这两个区域参与调控。突变体A55 U的结构探测，其中包含一个单核苷酸交换环L3的结果在一个改变的探测模式的环，但也对相反的凸起B1-2。这表示两个区域协作并形成复合结合袋。因此，我们的适体介导的翻译调控模型是无配体的适体对翻译起始只有轻微的影响。然后Tc以假结样方式导致分子内连接，并将适体转变为其抑制形式。这代表了一种新的核糖开关作用机制，与目前已知的天然核糖开关明显不同，天然核糖开关通过核糖体结合位点的螯合、转录衰减和核酶介导的降解发挥作用。
Riboswitches are newly discovered regulatory elements that consist solely of RNA, sense their ligand in a preformed binding pocket, and perform a conformational switch in response to ligand binding, resulting in altered gene expression. Regulation by a tetracycline (tc)-binding aptamer when inserted into the 5' untranslated region (UTR) of a reporter gene exhibits all characteristics of a riboswitch. Chemical and enzymatic probing reveals that the aptamer consists of two stems, P1 and P2, which are already present in the absence of tc and form the scaffold of the aptamer. They are separated by a bulge B1-2 and an opposing stem-loop (P3-L3). Tc-dependent changes in the probing pattern only appear in the upper part of the bulge B1-2 (nucleotides 9-13) and the loop L3. Saturating mutagenesis corroborates the involvement of these two regions in regulation. Structural probing of the mutant A55U, which contains a single-nucleotide exchange in loop L3 results in a changed probing pattern of the loop, but also of the opposing bulge B1-2. This denotes that both regions cooperate and form a composite binding pocket. Thus, our model for aptamer-mediated translational regulation is that the ligand-free aptamer has only marginal influence on translational initiation. Tc then leads to an intramolecular connection in a pseudoknot-like manner and turns the aptamer into its inhibitory form. This represents a new mechanism for riboswitch action clearly distinguished from currently known naturally occurring riboswitches, which function by sequestration of the ribosomal binding site, transcriptional attenuation, and ribozyme-mediated degradation.