Search for: All records

The glmS ribozyme riboswitch, located in the 5′ untranslated region of the Bacillus subtilis glmS messenger RNA (mRNA), regulates cell wall biosynthesis through ligand-induced self-cleavage and decay of the glmS mRNA. Although self-cleavage of the refolded glmS ribozyme has been studied extensively, it is not known how early the ribozyme folds and self-cleaves during transcription. Here, we combine single-molecule fluorescence with kinetic modeling to show that self-cleavage can occur during transcription before the ribozyme is fully synthesized. Moreover, co-transcriptional folding of the RNA at a physiological elongation rate allows the ribozyme catalytic core to react without the downstream peripheral stability domain. Dimethyl sulfate footprinting further revealed how slow sequential folding favors formation of the native core structure through fraying of misfolded helices and nucleation of a native pseudoknot. Ribozyme self-cleavage at an early stage of transcription may benefit glmS regulation in B. subtilis, as it exposes the mRNA to exoribonuclease before translation of the open reading frame can begin. Our results emphasize the importance of co-transcriptional folding of RNA tertiary structure for cis-regulation of mRNA stability.

Small ribozymes such asOryza sativatwister spontaneously cleave their own RNA when the ribozyme folds into its active conformation. The coupling between twister folding and self-cleavage has been difficult to study, however, because the active ribozyme rapidly converts to product. Here, we describe the synthesis of a photocaged nucleotide that releases guanosine within microseconds upon photosolvolysis with blue light. Application of this tool toO. sativatwister achieved the spatial (75 µm) and temporal (≤30 ms) control required to resolve folding and self-cleavage events when combined with single-molecule fluorescence detection of the ribozyme folding pathway. Real-time observation of single ribozymes after photo-deprotection showed that the precleaved folded state is unstable and quickly unfolds if the RNA does not react. Kinetic analysis showed that Mg²⁺and Mn²⁺ions increase ribozyme efficiency by making transitions to the high energy active conformation more probable, rather than by stabilizing the folded ground state or the cleaved product. This tool for light-controlled single RNA folding should offer precise and rapid control of other nucleic acid systems.