Reaction and interaction dynamics of azobenzene-tethered DNA with T7 RNA polymerase

Reaction and interaction dynamics of azobenzene-tethered DNA (photoresponsive DNA) with T7 RNA polymerase (T7RNAP) were studied after photoisomerization of azobenzene from the cis- to trans-forms using the transient grating (TG) and time-resolved fluorescence polarization techniques. Two types of photoresponsive DNA were examined: AzoPBD, tethered at the protein binding site, and AzoTATA, tethered at the unwinding site. A diffusion change was observed after photoexcitation of cis-AzoPBD within 1 ms, and this change is explained in terms of a structural change from a bent to an extended conformation upon the cis-to-trans photoisomerization. The association and dissociation rates of cis- and trans-AzoPBD-T7RNAP complex were measured by the fluorescence polarization measurements, and the dissociation constants for cis- and trans-AzoPBD were respectively determined to be 3.9 mu M and 21 mu M. The result indicates that trans-AzoPBD is dissociated upon photoexcitation of cis-AzoPBD-T7RNAP complex. The efficient dissociation is mainly caused by a small association rate constant. The time-resolved diffusion measurement showed a conformational change with a time constant of 2.4 ms and a dissociation reaction with a slower rate, which depends on the concentration of T7RNAP. Although AzoTATA does not exhibit significant structural changes upon isomerization, a diffusion change was observed upon photoexcitation of AzoTATA-T7RNAP. The origin is attributed to changes from the unwound to closed states of AzoTATA, but AzoTATA does not dissociate from T7RNAP. These findings highlight a critical role of the azobenzene insertion position in modulating DNA-T7RNAP interaction dynamics, providing new insights into light-regulated transcription control.