Abstract
Transcriptional riboswitches modulate downstream gene expression by a tight coupling of ligand-dependent RNA folding kinetics with the rate of transcription. RNA folding pathways leading to functional ON and OFF regulation involve the formation of metastable states within well-defined sequence intervals during transcription. The kinetic requirements for the formation and preservation of these metastable states in the context of transcription remain unresolved. Here, we reversibly trap the previously defined regulatory relevant metastable intermediate of the Mesoplasma florum 2′-deoxyguanosine (2′dG)-sensing riboswitch using a photocaging-ligation approach, and monitor folding to its native state by real-time NMR in both presence and absence of ligand. We further determine transcription rates for two different bacterial RNA polymerases. Our results reveal that the riboswitch functions only at transcription rates typical for bacterial polymerases (10–50 nt s−1) and that gene expression is modulated by 40–50% only, while subtle differences in folding rates guide population ratios within the structural ensemble to a specific regulatory outcome.
Highlights
Transcriptional riboswitches modulate downstream gene expression by a tight coupling of ligand-dependent RNA folding kinetics with the rate of transcription
While refolding from PA interaction becomes metastable (PA–T) to PA–T conformation. To monitor antiterminator (PAT) is initiated at nucleotide 113, segment 113–121 only contains four weak base pairs within helix PT, and, refolding kinetics from PA–T to PAT are most accurately described at length 121 on average
To determine shifts in population ratios as transcription proceeds further, we present a ligationbased approach that provides access to reversible trapping of metastable states that form within the activation and repression pathway of ON function
Summary
Transcriptional riboswitches modulate downstream gene expression by a tight coupling of ligand-dependent RNA folding kinetics with the rate of transcription. PAT states adopted within the second transcription interval (in green) become metastable as strand H is transcribed (PAT–H) during synthesis of nts 139 to 144 in both, absence or presence of ligand. Within this short period during transcription, the terminator conformation PA–TH represents the most stable state. It follows that deciphering the change of kinetics of antiterminator formation (PAT) depending on the absence and presence of ligand represents the most important information regarding regulation
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