Structural basis of ribosomal RNA transcription regulation

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.

Structure of the bacteriophage PhiKZ non-virion RNA polymerase

“…One region of particular note is the unresolved region of gp123. The very proximal regions of this domain have a visually similar topology to eubacterial β Si1 ( 29 ), however no further residues can be assigned with confidence. We note that the presence of this domain would be consistent with an evolutionary origin in the proteobacteria, which is expected given the host spectra of ΦKZ and related bacteriophages.…”

Structural origins of Escherichia coli RNA polymerase open promoter complex stability

“…Thus despite the presence of UP elements in these promoters and in the ribosomal promoters rpsT P2 and rrnB P1, by the time RPo has formed, the DNA upstream of -45 is dynamic and the DNA binding mode of the second aCTD is heterogeneous (34)(35)(36)45). Superposition of the cryo-EM RPo structures here and published high resolution cryo-EM RPo complexes [rpsT P2 (34) and rrnB P1 (36)] revealed small to moderate differences in the conformation of the clamp and the blobe (SI Appendix, Table S3). With respect to lPR class I, the clamp and the blobe are more open in all other RPo.…”

“…Resulting structures often revealed strand disorder; to improve resolution, NTPs or short RNA primers were added, forming transcription initiation complexes (RPinit). Until very recently (33)(34)(35)(36), the structure of RPo was mostly inferred from these RPinit's formed at nonnative sequences/structures. Similarly, detailed biochemical studies of RPo formation exist for only a small handful of promoters.…”

Recognition of Streptococcal Promoters by the Pneumococcal SigA Protein

“…As illustrated in Figure 2B , the main difference between both proteins is that the region from residue N128 to residue A372 of the σ70 factor is absent in SigA. Such a region is important for promoter escape and hinders early elongation pausing in σ70-like sigma factors ( Shin et al, 2021 ). The second-best model from SWISS-MODEL used the B. subtilis SigA structure (PDB 7ckq), which due to flexibility of the domain 1.1 misses that region in the determined structure.…”

“…In the RNA polymerase holoenzyme, the negatively charged domain 1.1 (a mimicry of DNA negative charge) needs to be displaced from its initial position for the DNA-RNA polymerase complex to become transcriptionally competent. The displacement process takes place more easily at some promoters while behaving more inhibitory or difficult to move out at others, therefore it assists in the promoter selection process ( Paget, 2015 ; Fang et al, 2020 ; Shin et al, 2021 ).…”