State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan, Hubei, People's Republic of China.
PLoS One. 2013 May 10;8(5):e62960. doi: 10.1371/journal.pone.0062960. Print 2013.
In bacteria, both promoters and 5'-untranslated regions (5'-UTRs) of mRNAs play vital regulatory roles in gene expression. In this study, we identified 1203 active promoter candidates in Bacillus thuringiensis through analysis of the genome-wide TSSs based on the transcriptome data. There were 11 types of σ-factor and 34 types of transcription factor binding sites found in 723 and 1097 active promoter candidates, respectively. Moreover, within the 1203 transcriptional units (TUs), most (52%) of the 5'-UTRs were 10-50 nucleotides in length, 12.8% of the TUs had a long 5'-UTR greater than 100 nucleotides in length, and 16.3% of the TUs were leaderless. We then selected 20 active promoter candidates combined with the corresponding 5'-UTR DNA regions to screen the highly active promoter-5'-UTR DNA region complexes with different characteristics. Our results demonstrate that among the 20 selected complexes, six were able to exert their functions throughout the life cycle, six were specifically induced during the early-stationary phase, and four were specifically activated during the mid-stationary phase. We found a direct corresponding relationship between σ-factor-recognized consensus sequences and complex activity features: the great majority of complexes acting throughout the life cycle possess σ(A)-like consensus sequences; the maximum activities of the σ(F)-, σ(E)-, σ(G)-, and σ(K)-dependent complexes appeared at 10, 14, 16, and 22 h under our experimental conditions, respectively. In particular, complex Phj3 exhibited the strongest activity. Several lines of evidence showed that complex Phj3 possessed three independent promoter regions located at -251∼-98, -113∼-31, and -54∼+14, and that the 5'-UTR +1∼+118 DNA region might be particularly beneficial to both the stability and translation of its downstream mRNA. Moreover, Phj3 successfully overexpressed the active β-galactosidase and turbo-RFP, indicating that Phj3 could be a proper regulatory element for overexpression of proteins in B. thuringiensis. Therefore, our efforts contribute to molecular biology research and the biotechnological application of B. thuringiensis.
在细菌中，mRNA 的启动子和 5'-非翻译区（5'-UTR）在基因表达中都起着重要的调控作用。在这项研究中，我们通过基于转录组数据的全基因组 TSS 分析，鉴定了苏云金芽孢杆菌中的 1203 个活性启动子候选者。在 723 个和 1097 个活性启动子候选者中分别发现了 11 种σ因子和 34 种转录因子结合位点。此外，在 1203 个转录单元（TU）中，大多数（52%）5'-UTR 的长度为 10-50 个核苷酸，12.8%的 TU 具有大于 100 个核苷酸的长 5'-UTR，16.3%的 TU 没有 5'-UTR。然后，我们选择了 20 个活性启动子候选者，并结合相应的 5'-UTR DNA 区域，以筛选具有不同特征的高活性启动子-5'-UTR DNA 区域复合物。我们的结果表明，在 20 个选定的复合物中，有 6 个能够在整个生命周期中发挥作用，有 6 个在早期静止期特异性诱导，有 4 个在中期静止期特异性激活。我们发现σ因子识别的保守序列与复合物活性特征之间存在直接对应关系：绝大多数在整个生命周期中起作用的复合物具有 σ(A)-样保守序列；在我们的实验条件下，σ(F)-、σ(E)-、σ(G)-和 σ(K)-依赖性复合物的最大活性分别出现在 10、14、16 和 22 h。特别是，复合物 Phj3 表现出最强的活性。有几条证据表明，复合物 Phj3 拥有三个位于-251∼-98、-113∼-31 和-54∼+14 的独立启动子区域，并且 5'-UTR +1∼+118 DNA 区域可能特别有利于其下游 mRNA 的稳定性和翻译。此外，Phj3 成功表达了活性β-半乳糖苷酶和 turbo-RFP，表明 Phj3 可以作为苏云金芽孢杆菌中蛋白质过表达的合适调控元件。因此，我们的努力有助于分子生物学研究和苏云金芽孢杆菌的生物技术应用。
