Understanding gene regulation and transcriptional kinetics at the molecular level is critical for understanding cellular function. Synthetic riboswitches have been developed using self-cleaving ribozyme expression platforms, where binding of the ligand causes a shift between a catalytically active and inactive conformation, or vice versa. Here, we employed three-dimensional orbital tracking fluorescence cross-correlation spectroscopy (3DOT-FCCS) to monitor transcriptional activity by following fluorescently labeled pre-mRNA in living cells. Using synthetic RNA regulatory systems, we probed the dynamics of transcription termination and ligand-dependent RNA cleavage mediated by a theophylline-responsive ribozyme aptamer. By tagging an exogenous HBB reporter gene in HEK293 T-Rex cells, we observed that the presence of an active ribozyme reduced the dwell time of nascent transcripts. This effect is consistent with premature cleavage of the RNA from the DNA template, enabling quantitative estimation of transcription elongation, termination, and ribozyme cleavage rates across three distinct ribozymes in the presence and absence of theophylline ligand. We determined that an elongation rate of ∼25 bp/s corresponds to a termination time of ∼92 s for the inactive control ribozyme, while a fast-cleaving control ribozyme exhibited cleavage times below 10 s. These findings provide new insights into transcriptional regulation and RNA-based control mechanisms in eukaryotic systems and establish a framework for engineering synthetic genetic circuits. This research is supported by the National Institute of General Medical Sciences (Grants 2R15GM123446-02A1 and 3R15GM123446-02A1S1).
Nisha et al. (Sun,) studied this question.