Effects of DNA Topology on Transcription from rRNA Promoters in Bacillus subtilis.

Petra Sudzinová,Hana Šanderová,Martin Benda,Olga Ramaniuk,Milada Kambová,Libor Krásný

doi:10.3390/microorganisms9010087

Petra Sudzinová, Hana Šanderová + Show 4 more

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https://doi.org/10.3390/microorganisms9010087

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Abstract

The expression of rRNA is one of the most energetically demanding cellular processes and, as such, it must be stringently controlled. Here, we report that DNA topology, i.e., the level of DNA supercoiling, plays a role in the regulation of Bacillus subtilis σA-dependent rRNA promoters in a growth phase-dependent manner. The more negative DNA supercoiling in exponential phase stimulates transcription from rRNA promoters, and DNA relaxation in stationary phase contributes to cessation of their activity. Novobiocin treatment of B. subtilis cells relaxes DNA and decreases rRNA promoter activity despite an increase in the GTP level, a known positive regulator of B. subtilis rRNA promoters. Comparative analyses of steps during transcription initiation then reveal differences between rRNA promoters and a control promoter, Pveg, whose activity is less affected by changes in supercoiling. Additional data then show that DNA relaxation decreases transcription also from promoters dependent on alternative sigma factors σB, σD, σE, σF, and σH with the exception of σN where the trend is the opposite. To summarize, this study identifies DNA topology as a factor important (i) for the expression of rRNA in B. subtilis in response to nutrient availability in the environment, and (ii) for transcription activities of B. subtilis RNAP holoenzymes containing alternative sigma factors.

Highlights

Bacterial cells need to adapt to environmental changes
We show that besides [GTP], B. subtilis ribosomal RNA (rRNA) promoters are regulated by the level of their supercoiling, and we dissect the effects of supercoiling on the formation of closed and open complexes, thereby providing mechanistic insights into the process
By reverse transcription followed by quantitative PCR (RT-qPCR) we monitored the response of rrnB P1 and Pveg to novobiocin treatment, using the same conditions as in the previous experiment

Summary

Introduction

Bacterial cells need to adapt to environmental changes. In nutrient-rich environments, cells grow and divide rapidly, and this requires a large number of ribosomes to satisfy the need for new proteins. As the production of new ribosomes is energetically costly for the cell, it must be tightly regulated. The number of ribosomes in the cell is regulated mainly on the level of transcription initiation of ribosomal RNA (rRNA) [1]. Transcription initiation can be divided into several steps. When the RNA polymerase (RNAP) holoenzyme (the core RNAP subunits [α2ββω] in complex with a σ factor) binds to specific DNA sequences, promoters, it forms the closed complex where

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