Institute of Microbiology and Molecular Biology, University of Giessen, Giessen, Germany.
Chair of Molecular Infection Biology II, Institute of Molecular Infection Biology (IMIB), University of Würzburg, Würzburg, Germany.
RNA Biol. 2022 Jan;19(1):980-995. doi: 10.1080/15476286.2022.2110380.
In , the methionine biosynthesis genes and are preceded by S-adenosyl-L-methionine (SAM) riboswitches of the SAM-II class. Upon SAM binding, structural changes in the riboswitch were predicted to cause transcriptional termination, generating the sRNA RZ. By contrast, the riboswitch was predicted to regulate translation from an AUG1 codon. However, downstream of the riboswitch, we found a putative Rho-independent terminator and an in-frame AUG2 codon, which may contribute to regulation. We validated the terminator between AUG1 and AUG2, which generates the sRNA RA1 that is processed to RA2. Under high SAM conditions, the activities of the and promoters and the steady-state levels of the read-through and mRNAs were decreased, while the levels of the RZ and RA2 sRNAs were increased. Under these conditions, the sRNAs and the mRNAs were stabilized. Reporter fusion experiments revealed that the Shine-Dalgarno (SD) sequence in the riboswitch is required for translation, which, however, starts 74 nucleotides downstream at AUG2, suggesting a novel translation initiation mechanism. Further, the reporter fusion data supported the following model of RNA-based regulation: Upon SAM binding by the riboswitch, the SD sequence is sequestered to downregulate translation, while the mRNA is stabilized. Thus, the SAM-II riboswitches fulfil incoherent, dual regulation, which probably serves to ensure basal and mRNA levels under high SAM conditions. This probably helps to adapt to changing conditions and maintain SAM homoeostasis.
在 中，甲硫氨酸生物合成基因 和 之前有 S-腺苷-L-甲硫氨酸 (SAM) Ⅱ类 SAM 核糖开关。SAM 结合后，预测 核糖开关的结构变化会导致转录终止，产生 sRNA RZ。相比之下，预测 核糖开关调节从 AUG1 密码子开始的翻译。然而，在 核糖开关的下游，我们发现了一个推定的 Rho 独立终止子和一个框内 AUG2 密码子，这可能有助于 调节。我们验证了 AUG1 和 AUG2 之间的终止子，它产生了被加工成 RA2 的 sRNA RA1。在高 SAM 条件下， 和 启动子的活性以及通读 的 和 mRNA 的稳态水平降低，而 RZ 和 RA2 sRNA 的水平增加。在这些条件下，sRNA 和 mRNA 被稳定。报告基因融合实验表明， 核糖开关中的 Shine-Dalgarno (SD) 序列对于翻译是必需的，然而，翻译起始于 AUG2 下游 74 个核苷酸处的 AUG2，这表明存在一种新的翻译起始机制。此外，报告基因融合数据支持基于 RNA 的调节的以下模型：当核糖开关结合 SAM 时，SD 序列被隔离以下调 翻译，而 mRNA 被稳定。因此，SAM-II 核糖开关实现了非相干的双重调节，这可能有助于在高 SAM 条件下维持基础 和 mRNA 水平。这可能有助于适应不断变化的条件并维持 SAM 同型平衡。
