Chair for Microbial Ecology, TUM School of Life Sciences, Department of Molecular Life Sciences, Technical University of Munich, Freising, Germany.
ZIEL - Institute for Food & Health, Technical University of Munich, Freising, Germany.
BMC Microbiol. 2023 Aug 31;23(1):243. doi: 10.1186/s12866-023-02988-6.
Analysis of genome wide transcription start sites (TSSs) revealed an unexpected complexity since not only canonical TSS of annotated genes are recognized by RNA polymerase. Non-canonical TSS were detected antisense to, or within, annotated genes as well new intergenic (orphan) TSS, not associated with known genes. Previously, it was hypothesized that many such signals represent noise or pervasive transcription, not associated with a biological function. Here, a modified Cappable-seq protocol allows determining the primary transcriptome of the enterohemorrhagic E. coli O157:H7 EDL933 (EHEC). We used four different growth media, both in exponential and stationary growth phase, replicated each thrice. This yielded 19,975 EHEC canonical and non-canonical TSS, which reproducibly occurring in three biological replicates. This questions the hypothesis of experimental noise or pervasive transcription. Accordingly, conserved promoter motifs were found upstream indicating proper TSSs. More than 50% of 5,567 canonical and between 32% and 47% of 10,355 non-canonical TSS were differentially expressed in different media and growth phases, providing evidence for a potential biological function also of non-canonical TSS. Thus, reproducible and environmentally regulated expression suggests that a substantial number of the non-canonical TSSs may be of unknown function rather than being the result of noise or pervasive transcription.
对全基因组转录起始位点（TSS）的分析表明，RNA 聚合酶不仅识别注释基因的典型 TSS，还识别非典型 TSS。非典型 TSS 被检测到位于注释基因的反义链上或内部，以及新的基因间（孤儿）TSS，它们与已知基因没有关联。此前，人们假设许多这样的信号代表与生物学功能无关的噪声或普遍转录。在这里，我们使用了一种改良的 Cappable-seq 方案来确定肠出血性大肠杆菌 O157:H7 EDL933（EHEC）的原始转录组。我们使用了两种不同的生长培养基，分别在指数生长期和静止生长期进行实验，每个条件重复三次。这产生了 19975 个 EHEC 典型和非典型的 TSS，它们在三个生物学重复中可重复出现。这对实验噪声或普遍转录的假设提出了质疑。因此，我们在启动子上游发现了保守的启动子基序，表明存在正确的 TSS。5567 个典型 TSS 中有超过 50%，10355 个非典型 TSS 中有 32%到 47%在不同的培养基和生长阶段中表达水平不同，这为非典型 TSS 也具有潜在生物学功能提供了证据。因此，可重复和环境调节的表达表明，大量非典型 TSS 可能具有未知功能，而不是噪声或普遍转录的结果。
