scholarly journals The bacterial promoter spacer modulates promoter strength and timing by length, TG-motifs and DNA supercoiling sensitivity

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Carlo A. Klein ◽  
Marc Teufel ◽  
Carl J. Weile ◽  
Patrick Sobetzko
Keyword(s):  
Promoter Activity ◽  
Gc Content ◽  
Promoter Sequence ◽  
Dna Supercoiling ◽  
Promoter Strength ◽  
Time Resolved ◽  
Spacer Length ◽  
Sequence Composition ◽  
Promoter Architecture ◽  
Transcriptomics Data

AbstractTranscription, the first step to gene expression, is a central coordination process in all living matter. Besides a plethora of regulatory mechanisms, the promoter architecture sets the foundation of expression strength, timing and the potential for further regulatory modulation. In this study, we investigate the effects of promoter spacer length and sequence composition on strength and supercoiling sensitivity in bacteria. Combining transcriptomics data analysis and standardized synthetic promoter libraries, we exclude effects of specific promoter sequence contexts. Analysis of promoter activity shows a strong variance with spacer length and spacer sequence composition. A detailed study of the spacer sequence composition under selective conditions reveals an extension to the -10 region that enhances RNAP binding but damps promoter activity. Using physiological changes in DNA supercoiling levels, we link promoter supercoiling sensitivity to overall spacer GC-content. Time-resolved promoter activity screens, only possible with a novel mild treatment approach, reveal strong promoter timing potentials solely based on DNA supercoiling sensitivity in the absence of regulatory sites or alternative sigma factors.

scholarly journals More than a Spacer: The Bacterial Promoter Spacer Modulates Promoter Strength and Timing by Length, Sequence, TG-Motifs and DNA Supercoiling Sensitivity

2021 ◽  
Author(s):  
Carlo A. Klein ◽  
Marc Teufel ◽  
Carl J. Weile ◽  
Patrick Sobetzko
Keyword(s):  
Promoter Activity ◽  
Gc Content ◽  
Promoter Sequence ◽  
Dna Supercoiling ◽  
Promoter Strength ◽  
Time Resolved ◽  
Spacer Length ◽  
Sequence Composition ◽  
Promoter Architecture ◽  
Transcriptomics Data

Abstract Transcription, the first step to gene expression, is a central coordination process in all living matter. Besides a plethora of regulatory mechanisms, the promoter architecture sets the foundation of expression strength, timing and the potential for further regulatory modulation. In this study, we investigate the effects of promoter spacer length and sequence composition on strength and supercoiling sensitivity in bacteria. Combining transcriptomics data analysis and standardized synthetic promoter libraries, we exclude effects of specific promoter sequence contexts. Analysis of promoter activity shows a strong variance with spacer length and spacer sequence composition. A detailed study of the spacer sequence composition under selective conditions reveals a, RNAP binding enhancing but expression damping, extension to the –10 region. Using physiological changes in DNA supercoiling levels, we link promoter supercoiling sensitivity to overall spacer GC-content. Time-resolved promoter activity screens, only possible with a novel mild treatment approach, reveal strong promoter timing potentials solely based on DNA supercoiling sensitivity in the absence of regulatory sites or alternative sigma factors.

scholarly journals Mutational Analysis of the ompA Promoter from Flavobacterium johnsoniae

2007 ◽  
Vol 189 (14) ◽  
pp. 5108-5118 ◽  
Author(s):  
Shicheng Chen ◽  
Michael Bagdasarian ◽  
Michael G. Kaufman ◽  
Adam K. Bates ◽  
Edward D. Walker
Keyword(s):  
Promoter Activity ◽  
Mutational Analysis ◽  
Core Promoter ◽  
Gc Content ◽  
Pronounced Effect ◽  
Promoter Elements ◽  
Spacer Length ◽  
Promoter Sequences ◽  
E Coli ◽  
Flavobacterium Johnsoniae

ABSTRACT Sequences that mediate the initiation of transcription in Flavobacterium species are not well known. The majority of identified Flavobacterium promoter elements show homology to those of other members of the phylum Bacteroidetes, but not of proteobacteria, and they function poorly in Escherichia coli. In order to analyze the Flavobacterium promoter structure systematically, we investigated the −33 consensus element, −7 consensus element, and spacer length of the Flavobacterium ompA promoter by measuring the effects of site-directed mutations on promoter activity. The nonconserved sequences in the spacer region and in regions close to the consensus motifs were randomized in order to determine their importance for promoter activity. Most of the base substitutions in these regions caused large decreases in promoter activity. The optimal −33/−7 motifs (TTTG/TANNTTTG) were identical to Bacteroides fragilis σABfr consensus −33/−7 promoter elements but lacked similarity to the E. coli σ70 promoter elements. The length of the spacer separating the −33 and −7 motifs of the ompA promoter also had a pronounced effect on promoter activity, with 19 bp being optimal. In addition to the consensus promoter elements and spacer length, the GC content of the core promoter sequences had a pronounced effect on Flavobacterium promoter activity. This information was used to conduct a scan of the Flavobacterium johnsoniae and B. fragilis genomes for putative promoters, resulting in 188 hits in B. fragilis and 109 hits in F. johnsoniae.

scholarly journals Identification and characterization of sequence signatures in the Bacillus subtilis promoter Pylb for tuning promoter strength

2019 ◽  
Vol 42 (1) ◽  
pp. 115-124
Author(s):  
Jiangtao Xu ◽  
Xiaoqing Liu ◽  
Xiaoxia Yu ◽  
Xiaoyu Chu ◽  
Jian Tian ◽  
...  
Keyword(s):  
Promoter Activity ◽  
Fluorescent Protein ◽  
Gc Content ◽  
Organophosphorus Hydrolase ◽  
Promoter Strength ◽  
Green Fluorescent ◽  
Mutant Promoter ◽  
Identification And Characterization ◽  
Sequence Signatures

Abstract Objective To thoroughly characterize the Pylb promoter and identify the elements that affect the promoter activity. Result The sequences flanking the − 35 and − 10 box of the Pylb promoter were divided into six segments, and six random-scanning mutant promoter libraries fused to an enhanced green fluorescent protein EGFP were made and analyzed by flow cytometry. Our results showed that the four nucleotides flanking the − 35 box could mostly influence the promoter activity, and this influence was related to the GC content. The promoters mutated in these regions were successfully used for expressing the gene ophc2 encoding organophosphorus hydrolase (OPHC2) and the gene katA encoding catalase (KatA). Conclusion Our work identified and characterized the sequence signatures of the Pylb promoter that could tune the promoter strength, providing further information for the potential application of this promoter. Meanwhile, the sequence signatures have the potential to be used for tuning gene expression in enzyme production, metabolic engineering, and synthetic biology.

scholarly journals Analysis of Promoters Recognized by HrpL, an Alternative σ-Factor Protein from Pantoea agglomerans pv. gypsophilae

2005 ◽  
Vol 18 (7) ◽  
pp. 634-643 ◽  
Author(s):  
Gal Nissan ◽  
Shulamit Manulis ◽  
Dan M. Weinthal ◽  
Guido Sessa ◽  
Isaac Barash
Keyword(s):  
Promoter Activity ◽  
Mutational Analysis ◽  
Present Report ◽  
Pantoea Agglomerans ◽  
Computational Techniques ◽  
Promoter Strength ◽  
Significant Activity ◽  
Gall Formation ◽  
Consensus Motif ◽  
Σ Factor

HrpL, an alternative σ factor, activates the transcription of the Hrp regulon by its binding to a common “hrp box” promoter. Based on computational techniques, the hrp box previously was defined as a consensus bipartite cis element, 5′-GGAACC-N15–16-CCACNNA-3′. The present report combines a quantitative in vivo assay for measuring Hrp promoter activity with site-specific mutagenesis to analyze the effect of consensus and nonconsensus nucleotides on promoter activity. The analysis was carried out with Hop effectors of the tumorigenic bacterium Pantoea agglomerans pv. gypsophilae, in which HrpL is indispensable for gall formation. Mutational analysis indicates that the hrp box consensus can be divided into crucial and noncrucial nucleotides. The first 5 nucleotides (nt) of the -35 consensus motif (GGAAC) and the 3 nt of the -10 motif (ACNNA) are crucial, whereas other consensus and adjacent nonconsensus nucleotides exert a significant effect on the promoter's strength. With spacing of 13 or 17 nt between the two motifs, significant activity was still retained. Gel shift assays indicated that deletion of GG from the -35 consensus motif eliminated HrpL binding, whereas mutations in the -10 consensus motif or modification of the spacing, which eliminates promoter activity, did not elicit any effect. The degeneracy in Hrp promoters of four hrp and type III effector genes of P. agglomerans pv. gypsophilae indicated significant differences in promoter activity, whereas increasing the promoter strength of the Hop effector, HsvG, resulted in overexpression of gall formation.

scholarly journals Transcription start site analysis reveals widespread divergent transcription in D. melanogaster and core promoter-encoded enhancer activities

2017 ◽  
Author(s):  
Sarah Rennie ◽  
Maria Dalby ◽  
Marta Lloret-Llinares ◽  
Stylianos Bakoulis ◽  
Christian Dalager Vaagensø ◽  
...  
Keyword(s):  
Transcription Initiation ◽  
Core Promoter ◽  
Regulatory Element ◽  
Regulatory Elements ◽  
Chromatin Interaction ◽  
Promoter Strength ◽  
Gene Promoters ◽  
Core Promoters ◽  
Promoter Architecture ◽  
Regulatory Functions

ABSTRACTMammalian gene promoters and enhancers share many properties. They are composed of a unified promoter architecture of divergent transcripton initiation and gene promoters may exhibit enhancer function. However, it is currently unclear how expression strength of a regulatory element relates to its enhancer strength and if the unifying architecture is conserved across Metazoa. Here we investigate the transcription initiation landscape and its associated RNA decay in D. melanogaster. Surprisingly, we find that the majority of active gene-distal enhancers and a considerable fraction of gene promoters are divergently transcribed. We observe quantitative relationships between enhancer potential, expression level and core promoter strength, providing an explanation for indirectly related histone modifications that are reflecting expression levels. Lowly abundant unstable RNAs initiated from weak core promoters are key characteristics of gene-distal developmental enhancers, while the housekeeping enhancer strengths of gene promoters reflect their expression strengths. The different layers of regulation mediated by gene-distal enhancers and gene promoters are also reflected in chromatin interaction data. Our results suggest a unified promoter architecture of many D. melanogaster regulatory elements, that is universal across Metazoa, whose regulatory functions seem to be related to their core promoter elements.

scholarly journals Influence of cis Element Arrangement on Promoter Strength in Trichoderma reesei

2017 ◽  
Vol 84 (1) ◽  
Author(s):  
Daniel P. Kiesenhofer ◽  
Robert L. Mach ◽  
Astrid R. Mach-Aigner
Keyword(s):  
Trichoderma Reesei ◽  
Transcription Start Site ◽  
Binding Sites ◽  
Inverted Repeat ◽  
Promoter Activity ◽  
Promoter Strength ◽  
Start Site ◽  
Transcription Start ◽  
Content Type ◽  
Sheer Number

ABSTRACTTrichoderma reeseican produce up to 100 g/liter of extracellular proteins. The major and industrially relevant products are cellobiohydrolase I (CBHI) and the hemicellulase XYNI. The genes encoding both enzymes are transcriptionally activated by the regulatory protein Xyr1. The first 850 nucleotides of thecbh1promoter contain 14 Xyr1-binding sites (XBS), and 8 XBS are present in thexyn1promoter. Some of these XBS are arranged in tandem and others as inverted repeats. One suchciselement, an inverted repeat, plays a crucial role in the inducibility of thexyn1promoter. We investigated the impact of the properties of suchciselements by shuffling them by insertion, exchange, deletion, and rearrangement ofciselements in both thecbh1andxyn1promoter. A promoter-reporter assay using theAspergillus nigergoxAgene allowed us to measure changes in the promoter strength and inducibility. Most strikingly, we found that an inverted repeat of XBS causes an important increase incbh1promoter strength and allows induction by xylan or wheat straw. Furthermore, evidence is provided that the distances ofciselements to the transcription start site have important influence on promoter activity. Our results suggest that the arrangement and distances ofciselements have large impacts on the strength of thecbh1promoter, whereas the sheer number of XBS has only secondary importance. Ultimately, the biotechnologically importantcbh1promoter can be improved byciselement rearrangement.IMPORTANCEIn the present study, we demonstrate that the arrangement ofciselements has a major impact on promoter strength and inducibility. We discovered an influence on promoter activity by the distances ofciselements to the transcription start site. Furthermore, we found that the configuration ofciselements has a greater effect on promoter strength than does the sheer number of transactivator binding sites present in the promoter. Altogether, the arrangement ofciselements is an important factor that should not be overlooked when enhancement of gene expression is desired.

Peptide nucleic acid restores colistin susceptibility through modulation of MCR-1 expression in Escherichia coli

2020 ◽  
Author(s):  
Xiaoming Wang ◽  
Yao Wang ◽  
Zhuoren Ling ◽  
Chaoyang Zhang ◽  
Mingming Fu ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Peptide Nucleic Acid ◽  
Promoter Activity ◽  
Sequence Variation ◽  
Promoter Sequence ◽  
E Coli ◽  
Expression In Escherichia Coli ◽  
Expression Host ◽  
Antisense Approach ◽  
Increased Susceptibility

Abstract Background Plasmid-mediated mechanisms of drug resistance accelerate the spread of polymyxin resistance, leaving clinicians with few or no antibacterial options for the treatment of infections caused by MDR bacteria, especially carbapenemase-producing strains. Objectives To evaluate the associations among promoter sequence variation, mcr-1 expression, host factors and levels of colistin resistance and to propose antisense agents such as peptide nucleic acids (PNAs) targeting mcr-1 as a tool to restore colistin susceptibility through modulation of MCR-1 expression in Escherichia coli. Methods A β-galactosidase assay was performed to study mcr-1 promoter activity. Quantitative real-time PCR and western blot assays were used to identify the expression level of MCR-1 in WT strains and transformants. Three PNAs targeting different regions of mcr-1 were designed and synthesized to determine whether they can effectively inhibit MCR-1 expression. MIC was measured to test colistin susceptibility in the presence or absence of PNA-1 in mcr-1-carrying E. coli. Results Variation in the mcr-1 promoter sequence and host species affect promoter activity, MCR-1 expression levels and colistin MICs. One PNA targeting the ribosome-binding site fully inhibited the expression of mcr-1 at a concentration of 4 μM, resulting in significantly increased susceptibility to colistin. The MIC90 of colistin decreased from 8 to 2 mg/L (P < 0.05) in the presence of 4 μM PNA. Conclusions These findings suggest that the antisense approach is a possible strategy to combat mcr-1-mediated resistance as well as other causes of emerging global resistance.

scholarly journals Promoter architecture dictates cell-to-cell variability in gene expression

Science ◽  
2014 ◽  
Vol 346 (6216) ◽  
pp. 1533-1536 ◽  
Author(s):  
Daniel L. Jones ◽  
Robert C. Brewster ◽  
Rob Phillips
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Messenger Rna ◽  
Experimental Studies ◽  
Promoter Strength ◽  
Promoter Architecture ◽  
Copy Numbers ◽  
Regulatory Architecture ◽  
Genome Wide ◽  
Cell To Cell Variability

Variability in gene expression among genetically identical cells has emerged as a central preoccupation in the study of gene regulation; however, a divide exists between the predictions of molecular models of prokaryotic transcriptional regulation and genome-wide experimental studies suggesting that this variability is indifferent to the underlying regulatory architecture. We constructed a set of promoters in Escherichia coli in which promoter strength, transcription factor binding strength, and transcription factor copy numbers are systematically varied, and used messenger RNA (mRNA) fluorescence in situ hybridization to observe how these changes affected variability in gene expression. Our parameter-free models predicted the observed variability; hence, the molecular details of transcription dictate variability in mRNA expression, and transcriptional noise is specifically tunable and thus represents an evolutionarily accessible phenotypic parameter.

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