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

Carlo A Klein,Patrick Sobetzko,Marc Teufel,Carl J Weile

doi:10.1038/s41598-021-03817-4

Carlo A Klein, Patrick Sobetzko + Show 2 more

Open Access

https://doi.org/10.1038/s41598-021-03817-4

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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 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.

Highlights

Transcription, the first step to gene expression, is a central coordination process in all living matter
By nutrient and stress-dependent changes in nucleoid-associated proteins (NAPs) abundance and topoisomerase activity, DNA supercoiling levels change throughout the bacterial growth c ycle[19]
We show that spacer length and sequence composition affect DNA supercoiling sensitivity and promoter strength significantly

Summary

Introduction

Transcription, the first step to gene expression, is a central coordination process in all living matter. We investigate the effects of promoter spacer length and sequence composition on strength and supercoiling sensitivity in bacteria. 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. All transcriptional regulation relies on the basal features of the promoter that are in turn modulated in strength and timing by additional regulatory mechanisms coupled to internal and external requirements. The basal promoter structure is interwoven with additional regulatory sites to bind proteins that modulate expression strength and timing. By nutrient and stress-dependent changes in NAP abundance and topoisomerase activity, DNA supercoiling levels change throughout the bacterial growth c ycle[19]. We show that spacer length and sequence composition affect DNA supercoiling sensitivity and promoter strength significantly. Using selective conditions, we identify novel sequence motifs within the spacer sequence

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