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

Abstract

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 μM and 21 μM. 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.