Translational activation by an alternative sigma factor in Bacillus subtilis. Translational activation by an alternative sigma factor in Bacillus subtilis

Sigma factors are an important class of bacterial transcription factors that lend specificity to RNA polymerases by binding to distinct promoter elements for genes in their regulons. Here we show that activation of the general stress sigma factor, B, in Bacillus subtilis paradoxically leads to dramatic induction of translation for a subset of its regulon genes. These genes are translationally repressed when transcribed by the housekeeping sigma factor, A, owing to extended RNA secondary structures as determined in vivo using DMS-MaPseq. Transcription fromB-dependent promoters liberates the secondary structures and activates translation, leading to dual induction. Translation efficiencies between B- and A-dependent RNA isoforms can vary by up to 100-fold, which in multiple cases exceeds the magnitude of transcriptional induction. These results highlight the role of long-range RNA folding in modulating translation and demonstrate that a transcription factor can regulate protein synthesis beyond its effects on transcript levels. Overall design: RNA was isolated from BS168 cells treated with DMS in vivo and from untreated cells. Regions of the σA-dependent isoforms of ctc and yvrE were specifically reverse-transcribed from each sample using TGIRT-III, which encoded DMS-induced RNA modifications as mutations in the resulting DNA amplicons. Libraries were prepared for sequencing following the established DMS-MaPseq protocol from Tomezsko et al., Nature, 2020 and Zubradt et al., Nature Methods, 2016. DMS signals were quantified using the DREEM algorithm from Tomezsko et al., Nature, 2020 and used as folding constraints to determine the RNA structure for each isoform.

σ因子（Sigma factors）是一类重要的细菌转录因子，可通过结合其调控子（regulon）内基因的特异性启动子元件，赋予RNA聚合酶转录特异性。本研究揭示，枯草芽孢杆菌（Bacillus subtilis）中普遍应激σ因子σB的激活，竟会反常地显著诱导其调控子内部分基因的翻译过程。这些基因在由持家σ因子（housekeeping sigma factor）σA介导转录时，会因延伸的RNA二级结构受到翻译抑制，该结论通过体内DMS-MaPseq技术得以验证。由σB依赖型启动子启动的转录可解除此类RNA二级结构的抑制作用并激活翻译，从而实现转录与翻译的双重诱导。σB与σA依赖型RNA异构体（RNA isoform）之间的翻译效率差异最高可达100倍，在多个案例中该差异幅度甚至超过了转录诱导的程度。上述结果凸显了长程RNA折叠在调控翻译过程中的关键作用，并证实转录因子可通过不依赖转录本水平的途径调控蛋白质合成。

实验设计概述：从经体内DMS处理与未处理的BS168菌株细胞中分离总RNA。针对每个样本，使用TGIRT-III逆转录酶特异性逆转录ctc与yvrE的σA依赖型RNA异构体区域，该逆转录过程会将DMS诱导的RNA修饰以突变形式体现在后续获得的DNA扩增子中。参照Tomezsko等于2020年发表于《自然（Nature）》、Zubradt等于2016年发表于《自然方法学（Nature Methods）》的DMS-MaPseq标准实验流程，构建测序文库。利用Tomezsko等于2020年《自然（Nature）》发表的DREEM算法（DREEM algorithm）对DMS信号进行定量，并将其作为折叠约束条件，确定每种RNA异构体的空间二级结构。