High-Throughput Identification of Promoters and Screening of Highly Active Promoter-5′-UTR DNA Region with Different Characteristics from Bacillus thuringiensis

PLOS ONE, Dec 2019

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 σA-like consensus sequences; the maximum activities of the σF-, σE-, σG-, and σK-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.

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High-Throughput Identification of Promoters and Screening of Highly Active Promoter-5′-UTR DNA Region with Different Characteristics from Bacillus thuringiensis

et al. (2013) High-Throughput Identification of Promoters and Screening of Highly Active Promoter-59-UTR DNA Region with Different Characteristics from Bacillus thuringiensis. PLoS ONE 8(5): e62960. doi:10.1371/journal.pone.0062960 High-Throughput Identification of Promoters and Screening of Highly Active Promoter-59-UTR DNA Region with Different Characteristics from Bacillus thuringiensis Jieping Wang. 0 Xulu Ai. 0 Han Mei. 0 Yang Fu 0 Bo Chen 0 Ziniu Yu 0 Jin He 0 Dipankar Chatterji, Indian Institute of Science, India 0 State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University , Wuhan, Hubei , People's Republic of China In bacteria, both promoters and 59-untranslated regions (59-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 s-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 59-UTRs were 10-50 nucleotides in length, 12.8% of the TUs had a long 59-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 59-UTR DNA regions to screen the highly active promoter-59-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 midstationary phase. We found a direct corresponding relationship between s-factor-recognized consensus sequences and complex activity features: the great majority of complexes acting throughout the life cycle possess sA-like consensus sequences; the maximum activities of the sF-, sE-, sG-, and sK-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 2251,298, 2113,231, and 254,+14, and that the 59-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 b-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. - Funding: This work was supported by the Chinese National Natural Science Funds (grants 31270105 and 30930004), the National Basic Research Program of China (973 Program, grant 2010CB126105), the National High-tech R&D Program of China (No. 2011AA10A205) and the Fundamental Research Funds for Central Universities of China (grant 2011PY092). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. Unlike archaea and eukaryotes, bacteria contain only one form of RNA polymerase (RNAP) core enzyme comprised of five subunits (a2bb9v). However, bacteria possess multiple forms of a specific s subunit (s-factor) and thus multiple forms of RNAP holoenzymes, which, in turn, bind to their cognate promoters to initiate transcription of specific genes (or operons) [14]. In bacteria, a promoter is a specific DNA sequence that provides secure initial binding sites for RNAP to initiate transcription of a particular gene (or operon) [1,2]. The core promoter includes a transcription start site (TSS) and two hexameric elements centered at or near 10 and 35 positions relative to the TSS. Some promoters contain one or more upstream promoter (UP) elements and the TGn extended 10 element, among others [14]. A TSS is an important marker of an active promoter, and mapping the TSSs is therefore a novel and effective strategy for the identification of active promoters. McGrath et al. mapped 769 TSSs and subsequently identified 27 promoter motifs in Caulobacter crescentus using a high-density array that was specifically designed to detect the TSS positions [5]. Mendoza-Vargas et al. mapped more than 1700 TSSs and identified a large number of promoters that control the expression of approximately 800 genes in Escherichia coli by combining a modified 59 RACE protocol and an unbiased high-throughput pyrosequencing strategy [6]. However, the active promoter candidates acquired by them were not verified through further experimentation. Recently, (...truncated)


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Jieping Wang, Xulu Ai, Han Mei, Yang Fu, Bo Chen, Ziniu Yu, Jin He. High-Throughput Identification of Promoters and Screening of Highly Active Promoter-5′-UTR DNA Region with Different Characteristics from Bacillus thuringiensis, PLOS ONE, 2013, Volume 8, Issue 5, DOI: 10.1371/journal.pone.0062960