Structural and Functional Insight into the Mechanism of Bacillus subtilis 6S-1 RNA Release from RNA Polymerase

Abstract

Here we investigated the refolding of Bacillus subtilis 6S-1 RNA and its release from σA-RNA polymerase (σA-RNAP) in vitro using truncated and mutated 6S-1 RNA variants. Truncated 6S-1 RNAs, only consisting of the central bubble (CB) flanked by two short helical arms, can still traverse the mechanistic 6S RNA cycle in vitro despite ~10-fold reduced σA-RNAP affinity. This indicates that the RNA’s extended helical arms including the ‘−35′-like region are not required for basic 6S-1 RNA functionality. The role of the ‘central bubble collapse helix’ (CBCH) in pRNA-induced refolding and release of 6S-1 RNA from σA-RNAP was studied by stabilizing mutations. This also revealed base identities in the 5’-part of the CB (5’-CB), upstream of the pRNA transcription start site (nt 40), that impact ground state binding of 6S-1 RNA to σA-RNAP. Stabilization of the CBCH by the C44/45 double mutation shifted the pRNA length pattern to shorter pRNAs and, combined with a weakened P2 helix, resulted in more effective release from RNAP. We conclude that formation of the CBCH supports pRNA-induced 6S-1 RNA refolding and release. Our mutational analysis also unveiled that formation of a second short hairpin in the 3′-CB is detrimental to 6S-1 RNA release. Furthermore, an LNA mimic of a pRNA as short as 6 nt, when annealed to 6S-1 RNA, retarded the RNA’s gel mobility and interfered with σA-RNAP binding. This effect incrementally increased with pLNA 7- and 8-mers, suggesting that restricted conformational flexibility introduced into the 5’-CB by base pairing with pRNAs prevents 6S-1 RNA from adopting an elongated shape. Accordingly, atomic force microscopy of free 6S-1 RNA versus 6S-1:pLNA 8- and 14-mer complexes revealed that 6S-1:pRNA hybrid structures, on average, adopt a more compact structure than 6S-1 RNA alone. Overall, our findings also illustrate that the wild-type 6S-1 RNA sequence and structure ensures an optimal balance of the different functional aspects involved in the mechanistic cycle of 6S-1 RNA.

Probing the conformational changes of in vivo overexpressed cell cycle regulator 6S ncRNA

“…The presence of pRNA molecules completely changes the conformation of the central domain, swelling it and making it more unstable and, ultimately, most likely incompatible with the RNAP cavity. These observations are also in agreement with an AFM analysis in Bacillus subtilis (Ganapathy et al, 2022), hinting at a universal mechanism of 6S conformational change and release. Moreover, the conformational shift of the 6S:pRNA complex occurred even with a modest molar excess of pRNA (1:3) and "survived" the passage through a size exclusion chromatography column inline with SAXS to separate from excess pRNA.…”

“…We present here for the first time, the 3D structure of the 6S:pRNA complex using models compatible with SAXS data obtained from Molecular Dynamics calculations. Our models not only are consistent with previous studies on 6S and 6S:pRNA complex ( Fadouloglou et al, 2015 ; Köhler et al, 2015 ; Ganapathy et al, 2022 ) but they also account for the flexibility of the nucleic acid particles and provide biophysical evidence for the structural rearrangements that pRNA synthesis induces to 6S and drive its release from RNAP ( Chen et al, 2017 ). Therefore, our work complements and expands on the present knowledge of the molecular mechanisms that govern the gene regulation through non-coding RNAs.…”

“…In summary, we provide evidence by Small Angle X-ray Scattering analysis that complex formation between E. coli 6S RNA and pRNA is associated with a compaction of the overall 6S size and an expansion of its central domain bulge. Similar observations for the Bacillus subtilis 6S-1 RNA ( Ganapathy et al, 2022 ) indicate that structural rearrangements induced by pRNA synthesis to 6S RNA may be a common property of non-coding 6S RNA cell cycle regulators and suggest a simple, elegant and universal mechanism of transcription control based on the abundance of nucleotides. The importance of this structural switch could be further supplemented by future experimental work on the stability and dynamics of the Eσ 70 :6S complexation, in the presence and absence of pRNA.…”

“…We often tend to think of RNA as a two-dimensional molecule and consequently few attempts have been made for a more comprehensive analysis of 6S with structural methods. These include the characterization of mature and precursor E. coli 6S with biophysical methods ( Fadouloglou et al, 2015 ), the Nuclear Magnetic Resonance analysis of Aquiflex aeolicus 6S ( Köhler et al, 2015 ), the cryo-electron microscopy study of the complex of E. coli Eσ 70 with 6S ( Chen et al, 2017 ) and the recent work on the Bacillus subtilis 6S with Atomic Force Microscopy (AFM) ( Ganapathy et al, 2022 ).…”