| Autor: |
Ganapathy S; Institut für Pharmazeutische Chemie, Philipps-Universität Marburg, 35037 Marburg, Germany., Hoch PG; Institut für Pharmazeutische Chemie, Philipps-Universität Marburg, 35037 Marburg, Germany., Lechner M; Zentrum für Synthetische Mikrobiologie, Philipps-Universität Marburg, 35032 Marburg, Germany., Bussiek M; Institute of Biology, University of Kassel, 34132 Kassel, Germany., Hartmann RK; Institut für Pharmazeutische Chemie, Philipps-Universität Marburg, 35037 Marburg, Germany. |
| Jazyk: |
angličtina |
| Zdroj: |
Non-coding RNA [Noncoding RNA] 2022 Feb 16; Vol. 8 (1). Date of Electronic Publication: 2022 Feb 16. |
| DOI: |
10.3390/ncrna8010020 |
| Abstrakt: |
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 '-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 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 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 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. |
| Databáze: |
MEDLINE |
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