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Abstract：
A riboswitch generally regulates the expression of its downstream genes through conformational change in its expression platform (EP) upon ligand binding. The cyclic diguanosine monophosphate (c-di-GMP) class I riboswitch Bc1 is widespread and conserved among Bacillus cereus group species. In this study, we revealed that Bc1 has a long EP with two typical ρ-independent terminator sequences 28 bp apart. The upstream terminator T1 is dominant in vitro, while downstream terminator T2 is more efficient in vivo. Through mutation analysis, we elucidated that Bc1 exerts a rare and incoherent \"transcription-translation\" dual regulation with T2 playing a crucial role. However, we found that Bc1 did not respond to c-di-GMP under in vitro transcription conditions, and the expressions of downstream genes did not change with fluctuation in intracellular c-di-GMP concentration. To explore this puzzle, we conducted SHAPE-MaP and confirmed the interaction of Bc1 with c-di-GMP. This shows that as c-di-GMP concentration increases, T1 unfolds but T2 remains almost intact and functional. The presence of T2 masks the effect of T1 unwinding, resulting in no response of Bc1 to c-di-GMP. The high Shannon entropy values of EP region imply the potential alternative structures of Bc1. We also found that zinc uptake regulator can specifically bind to the dual terminator coding sequence and slightly trigger the response of Bc1 to c-di-GMP. This work will shed light on the dual-regulation riboswitch and enrich our understanding of the RNA world.IMPORTANCEIn nature, riboswitches are involved in a variety of metabolic regulation, most of which preferentially regulate transcription termination or translation initiation of downstream genes in specific ways. Alternatively, the same or different riboswitches can exist in tandem to enhance regulatory effects or respond to multiple ligands. However, many putative conserved riboswitches have not yet been experimentally validated. Here, we found that the c-di-GMP riboswitch Bc1 with a long EP could form a dual terminator and exhibit non-canonical and incoherent \"transcription-translation\" dual regulation. Besides, zinc uptake regulator specifically bound to the coding sequence of the Bc1 EP and slightly mediated the action of Bc1. The application of SHAPE-MaP to the dual regulation mechanism of Bc1 may establish the foundation for future studies of such complex untranslated regions in other bacterial genomes.
摘要：
核糖开关通常在配体结合时通过其表达平台(EP)中的构象变化来调节其下游基因的表达。环状单磷酸二鸟苷（c-di-GMP）I类核糖开关Bc1在蜡状芽孢杆菌组物种中分布广泛且保守。在这项研究中,我们发现Bc1具有长EP，具有两个典型的ρ独立终止子序列，相距28bp。上游终止子T1在体外占优势，而下游终止子T2在体内更有效。通过突变分析,我们阐明了Bc1具有罕见且不连贯的“转录-翻译”双重调节，而T2起着至关重要的作用。然而,我们发现Bc1在体外转录条件下对c-di-GMP没有反应，下游基因的表达没有随着细胞内c-di-GMP浓度的波动而变化。为了探索这个谜题，我们进行了SHAPE-MaP，并确认了Bc1与c-di-GMP的相互作用。这表明，随着c-di-GMP浓度的增加，T1展开，但T2几乎保持完整和功能。T2的存在掩盖了T1退绕的影响，导致Bc1对c-di-GMP没有反应。EP区域的高Shannon熵值暗示了Bc1的潜在替代结构。我们还发现锌摄取调节剂可以特异性结合双终止子编码序列，并略微触发Bc1对c-di-GMP的反应。这项工作将阐明双重调节核糖开关，并丰富我们对RNA世界的理解。重要性质，核糖开关参与多种代谢调节，其中大多数优先以特定方式调节下游基因的转录终止或翻译起始。或者，相同或不同的核糖开关可以串联存在，以增强调节作用或响应多个配体。然而,许多假定的保守核糖开关尚未通过实验验证。这里,我们发现，具有长EP的c-di-GMP核糖开关Bc1可以形成双重终止子，并表现出非规范和不连贯的“转录-翻译”双重调节。此外,锌摄取调节剂特异性结合Bc1EP的编码序列，并略微介导Bc1的作用。将SHAPE-MaP应用于Bc1的双重调节机制可能为将来研究其他细菌基因组中此类复杂的非翻译区奠定基础。
