Systematic discovery of uncharacterized transcription factors in Escherichia coli K-12 MG1655 (RNA-seq data set)

Transcriptional regulation enables cells to respond to environmental changes. Yet, among the estimated 304 candidate transcription factors (TFs) in Escherichia coli K-12 MG1655, only 185 have been experimentally characterized. Here we developed an integrated workflow that contains the prediction of TFs using machine learning and comprehensive experimental validation using a suite of genome-wide experiments. Applying this workflow we: 1) computationally identified 16 candidate genes encoding uncharacterized TFs; 2) confirmed that ten of these 16 are TF candidates that showed 255 DNA binding sites; 3) found high-confidence TF-binding sequence motifs for six of the ten TFs; 4) reconstructed the regulons of the ten TFs by determining gene expression change upon deletion of each TF; and 5) further determined the regulatory roles of three TFs (YdcI, YeiE and YiaJ) to be regulating acetate metabolism, iron homeostasis under iron limited condition, and utilization of L-ascorbate, respectively. Together, these results demonstrate how the integrated workflow can be used to discover, characterize, and elucidate regulatory functions of uncharacterized TFs. This workflow can be applied to less studied bacteria for systematic discovery and characterization of their transcriptional regulatory networks. Overall design: Expression profiling data for four uncharacterized transcription factor deletion strains were generated by RNA-seq, in duplicate

转录调控（transcriptional regulation）可使细胞响应环境变化。然而在大肠杆菌K-12 MG1655预估的304个候选转录因子（transcription factors, TFs）中，仅185个已完成实验表征。本研究开发了一套整合式工作流，涵盖基于机器学习的转录因子预测方案，以及依托全基因组实验的综合验证策略。应用该工作流，我们完成了以下工作：1）通过计算鉴定出16个编码未表征转录因子的候选基因；2）证实该16个候选基因中有10个为转录因子候选物，共检测到255个DNA结合位点；3）为这10个转录因子中的6个找到了高可信度的转录因子结合序列基序；4）通过测定每个转录因子缺失后的基因表达变化，重构了这10个转录因子的调控子（regulon）；5）进一步明确了3个转录因子（YdcI、YeiE及YiaJ）的调控功能，分别为调控乙酸代谢、铁限制条件下的铁稳态，以及L-抗坏血酸的利用。综上，本研究结果证实该整合式工作流可用于发现、表征并阐明未表征转录因子的调控功能。该工作流可推广应用于研究程度较低的细菌，以系统性发现并表征其转录调控网络。整体实验设计：针对4株未表征转录因子缺失菌株的表达谱数据，通过RNA测序（RNA-seq）生成，且设置了两次生物学重复。