Stabilizing Obligatory Non-native Intermediates Along Co-transcriptional Folding Trajectories of SRP RNA Affects Cell Viability

Abstract

SummarySignal recognition particle (SRP) in Escherichia coli comprises protein Ffh and SRP RNA. Its essential functionality-co-translational protein-targeting/delivery to cellular membraneshinges on the RNA attaining a native long-hairpin fold that facilitates protein conformational rearrangements within the SRP complex. Since RNA folds co-transcriptionally on RNA polymerase, we use high-resolution optical tweezers to first characterize the mechanical unfolding/refolding of incrementally lengthened RNAs from stalled transcription complexes until reaching the full-length transcript. This analysis allows identification of folding intermediates adopted during the real-time co-transcriptional folding of SRP RNA. The cotranscriptional folding trajectories are surprisingly invariant to transcription rates, and involve formation of an obligatory non-native hairpin intermediate that eventually resolves into the native fold. SRP RNA variants designed to stabilize this non-native intermediate-likely sequestering the SRP ribonucleoprotein complex in an inactive form-greatly reduce cell viability, indicating that the same co-transcriptional folding mechanism operates in vivo and possible alternative antibiotic strategies.

DUETT quantitatively identifies known and novel events in nascent RNA structural dynamics from chemical probing data

“…This hairpin rearranges into the long helical structure, which DUETT identifies as downswing events between lengths 116 and 127 nt in bases 11, 14-15, and 17-19. These identifications are consistent with recent computational modeling (Yu et al, 2018) and single molecule optical tweezer experiments (Fukuda et al, 2018) that propose the rearrangement of H1 to occur in the window that DUETT detects.…”

“…Previous studies have shown that during transcription, the E.coli SRP RNA forms a transient 5' hairpin (H1) that rearranges into a long helical structure with a hairpin loop and multiple inner loops (Batey et al, 2000;Wong et al, 2007;Watters, Strobel, et al, 2016), which we label H2-H5 ( Figure 2). Several of these transitions have been validated by prior bulk studies (Wong et al, 2007;Watters, Strobel, et al, 2016), and by single molecule optical trapping experiments (Fukuda et al, 2018). A previous Here DUETT is applied to a mock dataset to identify an increase in reactivities in several consecutive nucleotides which is consistent with the typical 'low-high-low' pattern observed for RNA hairpin structures.…”

“…The major cotranscriptional rearrangement event for the E. coli SRP RNA occurs when H1 refolds into the final extended structure (Yu et al, 2018;Fukuda et al, 2018). Corresponding to the formation of H1, DUETT identified upswings in bases 14-15 and 18-19 around nascent RNA length 45 nt, and upramps that conclude around length 50 nt ( Figure 2).…”

“…Another key feature of the E. coli SRP RNA cotranscriptional folding pathway is the formation of native base pairs and loops after the formation of H1, but before the rearrangement of H1 into the final native structure (Wong et al, 2007;Watters, Strobel, et al, 2016;Fukuda et al, 2018;Yu et al, 2018). DUETT identifies expected reactivity signatures of bases 26-29 and 31 through length 100 nt.…”

A mechanism for ligand gated strand displacement in ZTP riboswitch transcription regulation

“…However, local off-pathway folds are unexpected for riboswitch folding pathways since function requires successful aptamer folding and therefore mandates that non-native intermediates be resolved efficiently. Despite this, we observed that pfl riboswitch folding involves formation of the temporary helix IH1, which precedes P1 folding, making this one of the few examples of non-native intermediate folds have been experimentally observed (Fukuda et al, 2018;Incarnato et al, 2017;Wong et al, 2007;Yu et al, 2018) (Figure 2). Interestingly, the IH1 structure is not explicitly encoded in the pfl aptamer consensus sequence.…”