Environment-dependent fluorescence intensity enhancement of Cy3 has also been exploited to study the formation of an unwound transcription initiation bubble comprised of ssDNA segments by RNA polymerase (RNAP) as it binds and unwinds promoter dsDNA. In a first ensemble-level study, Ko and Heyduk [100 (link)] reported that the fluorescence intensity from a Cy3 strategically placed on promoter DNA showed a ~two-fold increase upon binding of RNAP. Subsequently, the Cy3 signature showed a similar decrease after transcription initiation and promoter escape. The results and control experiments described in the same report indicated that the observed fluorescence intensity increase is due to the unwinding of dsDNA to ssDNA upon RNAP binding, while a decrease results from the rewinding of ssDNA to dsDNA upon promoter escape. The large ~two-fold fluorescence enhancement in unwinding-induced fluorescence enhancement (UIFE) assays could possibly result from a combination of binding of RNAP to the promoter dsDNA, unwinding of promoter dsDNA to ssDNA segments, and subsequent conformational changes involving the unwound ssDNA segment and RNAP. The ensemble assay is simple and straightforward to implement and has been used extensively in studies investigating the mechanism of promoter unwinding and promoter escape in transcription by several groups [62 (link),100 (link)–103 ].
Later, the Ha lab implemented a single-molecule UIFE (smUIFE) assay to study the kinetics and mechanism of transcription initiation by a phage T7 RNAP [104 (link)]. More recently, similar smUIFE experiments were used in real-time single-molecule assays investigating the promoter unwinding mechanism by a bacterial RNAP (Figure 4C ). Here, the authors monitored the unwinding kinetics of the upstream and downstream segments of a promoter fragment to show that unwinding occurs in steps that proceed from upstream towards the downstream direction [74 (link)]. The smUIFE assays can potentially be combined with high-throughput single-molecule studies of large promoter sequence libraries, enabling a complete dissection of the promoter sequence dependence during this stage of transcription initiation. Similar assays can also be used in other processes that involve DNA unwinding and rewinding, such as replication initiation and nucleic acid helicase and topoisomerase activities. Notably, such assays will carry different signal contributions from the unwinding and the rewinding process, as well as from the proximity of the protein machinery.
Importantly, fluorescence enhancement mechanisms similar to the ones mentioned above exist, where stabilization of the planar excited-state occurs via binding to a molecular scaffold, are used in other fluorescent probes. These dyes are useful due to their increased fluorescence upon binding to, e.g., nucleic acids (e.g., TOTO, YOYO) [105 (link)] or to amyloid-like fibrils (e.g., Thioflavin T [106 (link)], Nile red [107 ]).
Later, the Ha lab implemented a single-molecule UIFE (smUIFE) assay to study the kinetics and mechanism of transcription initiation by a phage T7 RNAP [104 (link)]. More recently, similar smUIFE experiments were used in real-time single-molecule assays investigating the promoter unwinding mechanism by a bacterial RNAP (
Importantly, fluorescence enhancement mechanisms similar to the ones mentioned above exist, where stabilization of the planar excited-state occurs via binding to a molecular scaffold, are used in other fluorescent probes. These dyes are useful due to their increased fluorescence upon binding to, e.g., nucleic acids (e.g., TOTO, YOYO) [105 (link)] or to amyloid-like fibrils (e.g., Thioflavin T [106 (link)], Nile red [107 ]).
