Kinetic proofreading as a mechanism for transcriptional specificity in living human cells

Abstract How target genes selectively respond to their specific transcription factors (TF) amid the vast excess of non-specific TFs in the nucleus remains a fundamental question in gene regulation. Here, we develop an integrated single-molecule imaging framework to quantitatively link TF dynamics with nascent transcription kinetics at endogenous gene loci to address the transcription factor specificity. Using endogenously Halo-tagged glucocorticoid receptor (GR), we show that ligand activation rapidly increases GR chromatin binding and residence times without substantially altering search kinetics. Live-cell nascent RNA imaging reveals that GR is essential for Dex induction of ERRFI1 transcription via increased burst frequency. Gene locus-specific, dual-color tracking demonstrates that GR display longer residence times in general near its target gene ERRFI1 compared to the non-target MYH9 locus, consistent with a kinetic proofreading model. A high-throughput imaging-based CRISPR screen identifies ATP-dependent processes, including neddylation and chromatin remodeling, and similarly, acute inhibition of the TFIIH XPB selectively impairs ERRFI1 transcription while sparing MYH9 , implicating ATPase activity in GR target discrimination. In all, these findings establish that promoters function as dwell-time rather than occupancy detectors, discriminating specific from non-specific TF interactions through energy-dependent mechanisms.