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

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...

Ausführliche Beschreibung

Gespeichert in:
Bibliographische Detailangaben
Autoren: Ganapathy, Sweetha, Hoch, Philipp G., Lechner, Marcus, Bussiek, Malte, Hartmann, Roland K.
Format: Artikel
Sprache:Englisch
Veröffentlicht: Philipps-Universität Marburg 2022
Schlagworte:
Bacillus subtilis 6S-1 RNA
truncated and mutated derivatives
Medizin
6S-1 RNA:σA-RNAP release kinetics
product RNA (pRNA) transcription
gel shift assays;
Medical sciences Medicine
Online Zugang:PDF-Volltext
Tags: Tag hinzufügen
Keine Tags, Fügen Sie den ersten Tag hinzu!
Beschreibung
Zusammenfassung: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.
Beschreibung:Gefördert durch den Open-Access-Publikationsfonds der UB Marburg.
Umfang:27 Seiten
DOI:10.3390/ncrna8010020

Ähnliche Einträge