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Systematic discovery of uncharacterized transcription factors in Escherichia coli K-12 MG1655

ABSTRACT: Systematic discovery of uncharacterized transcription factors in Escherichia coli K-12 MG1655

PROVIDER: PRJNA435923 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR6781980.fastq.gz	Fastqsanger.gz
	SRR6781990.fastq.gz	Fastqsanger.gz
	SRR6782000_1.fastq.gz	Fastqsanger.gz
	SRR6782000_2.fastq.gz	Fastqsanger.gz
	SRR6781981.fastq.gz	Fastqsanger.gz

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

Project description: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.

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Project description:Systematic discovery of uncharacterized transcription factors in Escherichia coli K-12 MG1655 (ChIP-exo data set)

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Systematic identification and characterization of uncharacterized transcription factors in Escherichia coli K-12 MG1655

Project description:Bacteria regulate gene expression to adapt to changing environments through transcriptional regulatory networks (TRNs). Although extensively studied, TRNs are not fully characterized since we do not know the identity of all the transcriptional regulators that comprise a TRN. Here, we experimentally evaluate 40 uncharacterized proteins in the model organisms Escherichia coli K-12 MG1655, which were previously predicted to be transcription factors (TFs). First, we used myc-tagging and a multiplexed ChIP-exo assay to characterize genome-wide binding sites for the 40 candidate TFs. Then, we compared those binding sites with those of RNA polymerase and sigma factors to increase the confidence in the measured DNA-binding events. Finally, we used these data to infer potential functional annotation for 13 candidate TFs. As a result, we characterized 570 new binding sites for 34 of 40 candidate TFs. Taken together, the study; 1) expands the number of confirmed TFs from 249 to 278, that is close to the estimated total of about 280 TFs in E. coli K-12 MG1655, 2) it provides a roadmap for functional characterization of the 34 discovered TFs, and 3) it enables the elucidation of the first comprehensive TRN.

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Systematic discovery of uncharacterized transcription factors in Escherichia coli K-12 MG1655.

Project description:Transcriptional regulation enables cells to respond to environmental changes. Of the estimated 304 candidate transcription factors (TFs) in Escherichia coli K-12 MG1655, 185 have been experimentally identified, but ChIP methods have been used to fully characterize only a few dozen. Identifying these remaining TFs is key to improving our knowledge of the E. coli transcriptional regulatory network (TRN). Here, we developed an integrated workflow for the computational prediction and comprehensive experimental validation of TFs using a suite of genome-wide experiments. We applied this workflow to (i) identify 16 candidate TFs from over a hundred uncharacterized genes; (ii) capture a total of 255 DNA binding peaks for ten candidate TFs resulting in six high-confidence binding motifs; (iii) reconstruct the regulons of these ten TFs by determining gene expression changes upon deletion of each TF and (iv) identify the regulatory roles of three TFs (YiaJ, YdcI, and YeiE) as regulators of l-ascorbate utilization, proton transfer and acetate metabolism, and iron homeostasis under iron-limited conditions, respectively. Together, these results demonstrate how this workflow can be used to discover, characterize, and elucidate regulatory functions of uncharacterized TFs in parallel.

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