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

Signal recognition particle (SRP) in Escherichia coli comprises protein Ffh and SRP RNA. Its essential functionality--co-translational protein-targeting/delivery to cellular membranes--hinges 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 co-transcriptional 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.