Structural basis of ribosomal RNA transcription regulation

Yeonoh Shin,Katsuhiko S Murakami,Andrey Kulbachinskiy,Daria Esyunina,Danil Pupov,M Zuhaib Qayyum

doi:10.1038/s41467-020-20776-y

Yeonoh Shin, Katsuhiko S Murakami + Show 4 more

Open Access

https://doi.org/10.1038/s41467-020-20776-y

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Abstract

Ribosomal RNA (rRNA) is most highly expressed in rapidly growing bacteria and is drastically downregulated under stress conditions by the global transcriptional regulator DksA and the alarmone ppGpp. Here, we determined cryo-electron microscopy structures of the Escherichia coli RNA polymerase (RNAP) σ70 holoenzyme during rRNA promoter recognition with and without DksA/ppGpp. RNAP contacts the UP element using dimerized α subunit carboxyl-terminal domains and scrunches the template DNA with the σ finger and β’ lid to select the transcription start site favorable for rapid promoter escape. Promoter binding induces conformational change of σ domain 2 that opens a gate for DNA loading and ejects σ1.1 from the RNAP cleft to facilitate open complex formation. DksA/ppGpp binding also opens the DNA loading gate, which is not coupled to σ1.1 ejection and impedes open complex formation. These results provide a molecular basis for the exceptionally active rRNA transcription and its vulnerability to DksA/ppGpp.

Highlights

Ribosomal RNA is most highly expressed in rapidly growing bacteria and is drastically downregulated under stress conditions by the global transcriptional regulator DksA and the alarmone ppGpp
Structural and biochemical studies of bacterial RNA polymerase (RNAP) transcription suggest that the order of DNA loading around the TSS and DNA opening may be interchangeable during promoter recognition (i.e., DNA melts first outside RNAP or DNA melts after loading inside the RNAP cleft) depending on σ factors, promoters, transcription factors and conditions[15,33,41,42]
We hypothesize that RNAP may use alternative mechanisms of rrnBP1 promoter open complex (RPo) formation with rrnBP1 and possibly other promoters, requiring the opening of the DNA loading gate, σ1.1 ejection from the DNA binding channel, and unwinding of the −10 element plus discriminator DNA, depending on the absence or presence of DksA/ppGpp

Summary

Introduction

Ribosomal RNA (rRNA) is most highly expressed in rapidly growing bacteria and is drastically downregulated under stress conditions by the global transcriptional regulator DksA and the alarmone ppGpp. The rate of ribosome biogenesis is primarily determined by rRNA transcription[1,2], which constitutes as much as 70% of total RNA synthesis and is initiated approximately every second from each of the seven rRNA operons (rrnA-E and rrnG-H) in E. coli during exponential growth[3] It is drastically repressed under stress conditions such as nutrient-starved stationary phase[4]. The G + C-rich discriminator and unusual TSS selection of rRNA promoters make its open complex (RPo) unstable, but facilitate RNAP escape from the promoter by reducing abortive RNA cycle prior to the RNA elongation stage[6] These promoter elements play key roles in the wide range of rRNA transcription regulation between nutrientrich and -poor growth conditions. These promoter elements play key roles in the wide range of rRNA transcription regulation between nutrientrich and -poor growth conditions. rRNA transcription activity is regulated by two small molecules—

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