ABSTRACT

Here, we describe SR7, a dual-function antisense RNA encoded on the Bacillus subtilis chromosome. This RNA was earlier described as SigB-dependent regulatory RNA S1136 and reported to reduce the amount of the small ribosomal subunit under ethanol stress. We found that the 5ʹ portion of SR7 encodes a small protein composed of 39 amino acids which we designated SR7P. It is translated from a 185 nt SigB-dependent mRNA under five different stress conditions and a longer SigB-independent RNA constitutively. About three-fold higher amounts of SR7P were detected in B. subtilis cells exposed to salt, ethanol, acid or heat stress. Co-elution experiments with SR7P_C-FLAG and Far-Western blotting demonstrated that SR7P interacts with the glycolytic enzyme enolase. Enolase is a scaffolding component of the B. subtilis degradosome where it interacts with RNase Y and phosphofructokinase PfkA. We found that SR7P increases the amount of RNase Y bound to enolase without affecting PfkA. RNA does not bridge the SR7P-enolase-RNase Y interaction. In vitro-degradation assays with the known RNase Y substrates yitJ and rpsO mRNA revealed enhanced enzymatic activity of enolase-bound RNase Y in the presence of SR7P. Northern blots showed a major effect of enolase and a minor effect of SR7P on the half-life of rpsO mRNA indicating a fine-tuning role of SR7P in RNA degradation.

摘要

在这里，我们描述了 SR7，一种在枯草芽孢杆菌染色体上编码的双功能反义 RNA 。这种 RNA 早先被描述为 SigB 依赖性调节 RNA S1136，据报道在乙醇压力下会减少核糖体小亚基的数量。我们发现 SR7 的 5ʹ 部分编码一种由 39 个氨基酸组成的小蛋白质，我们将其命名为 SR7P。它在五种不同的压力条件下由 185 nt SigB 依赖性 mRNA 和更长的 SigB 非依赖性 RNA 组成。在暴露于盐、乙醇、酸或热应激的枯草芽孢杆菌细胞中检测到的 SR7P 量大约高出三倍。使用 SR7P _{C-FLAG 进行}共洗脱实验Far-Western 印迹表明 SR7P 与糖酵解酶烯醇化酶相互作用。烯醇酶是枯草芽孢杆菌降解体的支架成分，在那里它与 RNase Y 和磷酸果糖激酶 PfkA 相互作用。我们发现 SR7P 在不影响 PfkA 的情况下增加了与烯醇化酶结合的 RNase Y 的量。RNA 不会桥接 SR7P-烯醇化酶-RNase Y 相互作用。用已知的 RNase Y 底物yitJ和rpsO mRNA 进行的体外降解试验揭示了在 SR7P 存在下与烯醇化酶结合的 RNase Y 的酶活性增强。Northern印迹显示烯醇化酶的主要影响和SR7P对rpsO mRNA半衰期的次要影响，表明SR7P在RNA降解中的微调作用。