Structural and Functional Insight into the Mechanism of Bacillus subtilis 6S-1 RNA Release from RNA Polymerase
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 m...
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| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Philipps-Universität Marburg
2022
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| Subjects: | |
| Online Access: | PDF Full Text |
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| 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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| Item Description: | Gefördert durch den Open-Access-Publikationsfonds der UB Marburg. |
| Physical Description: | 27 Pages |
| DOI: | 10.3390/ncrna8010020 |