Genetically Expanded Fluorescent SSBT7 RNA Polymerase in Action: Intriguing Photophysics with Enhanced RNA Production

T7 RNA polymerase (T7-RNAP) is a highly efficient transcriptional enzyme. However, it is prone to mutation at the functionally important Y571 residue, which strongly influences its functional properties. Here, we demonstrate a strategy to modulate the T7-RNAP enzyme by replacing the Y571 residue with a fluorescent triazolyl unnatural amino acid (FTUAA), TCNBAlaAc, via genetic code expansion (GCE) technology using two orthogonal aminoacyl-tRNA synthetase/tRNA pairs. Thus, TCNBAlaAc-encoded variants, herein termed SSBT7-RNAP, were successfully expressed, purified, and characterized by SDS-PAGE, MALDI-TOF mass spectrometry, CD, UV–vis, and fluorescence spectroscopy. Incorporation of TCNBAlaAc introduced distinct and enhanced fluorescence signatures while largely preserving the overall secondary structures of the engineered enzymes. Notably, these novel TCNBAlaAc-encoded variants exhibited higher relative RNA yields than those of wild-type T7-RNAP under identical in vitro transcription conditions. Molecular docking further supported the structural compatibility of the modification with the promoter-bound enzyme. These findings establish GCE-based, site-specific incorporation of an FTUAA into a functionally relevant site of T7 RNA polymerase as a promising strategy for generating spectroscopically traceable, transcriptionally competent T7-RNAP variants for future enzyme engineering and mechanistic studies.