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Titel:Structural and Functional Insight into the Mechanism of Bacillus subtilis 6S-1 RNA Release from RNA Polymerase
Autor:Ganapathy, Sweetha
Weitere Verfasser:Hoch, Philipp G.; Lechner, Marcus; Bussiek, Malte; Hartmann, Roland K.
Veröffentlicht:2022
URI:https://archiv.ub.uni-marburg.de/es/2022/0156
DOI: https://doi.org/10.3390/ncrna8010020
URN: urn:nbn:de:hebis:04-es2022-01561
DDC:610 Medizin
Publikationsdatum:2022-09-01
Lizenz:https://creativecommons.org/licenses/by/4.0

Dokument

Schlagwörter:
Bacillus subtilis 6S-1 RNA, truncated and mutated derivatives, 6S-1 RNA:σA-RNAP release kinetics, product RNA (pRNA) transcription, gel shift assays;

Summary:
Here we investigated the refolding of Bacillus subtilis 6S-1 RNA and its release from σ A-RNA polymerase (σ A-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 σ A-RNAP affinity. This indicates that the RNA’s extended helical arms including the ‘−350 -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 σ A-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 σ A-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 30 -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 σ A-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.


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