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.
Highlights
Bacterial 6S RNAs are non-coding RNAs of ~200 nt that form a rod-shaped secondary structure with a relatively unstructured region in the center that is flanked by two, non-continuously helical arms: the terminal stem formed by the RNA’s 50 - and 30 -proximal sequences and the internal stem capped by a loop (Figure 1 and Figure S1)
In B. subtilis 6S-1 RNA and A. aeolicus 6S RNA, another structural element was inferred to form upon product RNA (pRNA) transcription, termed the ‘central bubble collapse helix (CBCH)’, involving the 30 -strand of P2 and the distal sequence of the 50 -central bubble (50 -CB). These findings suggest differences in the pRNA-induced 6S RNA release mechanism of E. coliand B. subtilis-type 6S RNAs
Complex have led to the identification of structural 6S RNA elements that correspond to the −35 and −10 regions of DNA promoters and interact with σ70 as well as the β and β’ subunits of the core RNAP [14–16]
Summary
Bacterial 6S RNAs are non-coding RNAs (ncRNA) of ~200 nt that form a rod-shaped secondary structure with a relatively unstructured region in the center (termed central bubble) that is flanked by two, non-continuously helical arms: the terminal (closing) stem formed by the RNA’s 50 - and 30 -proximal sequences and the internal (apical) stem capped by a loop (Figure 1 and Figure S1). The sequence identity of 6S RNAs is limited and only the inclusion of secondary structure conservation (covariance models) allowed their identification as members of the same ncRNA family [1]. The sequence identity between Bacillus subtilis 6S-1 RNA and Escherichia coli 6S RNA is 38 to 45%, depending on. Non-coding RNA 2022, 8, 20 identity between Bacillus subtilis 6S-1 RNA and Escherichia coli 6S RNA is 38 to 45%, depending on the type of alignment (Figure S1B).
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