Abstract
In eukaryotic cells, RNA Polymerase II (RNAP2) is the enzyme in charge of transcribing mRNA from DNA. RNAP2 possesses an extended carboxy-terminal domain (CTD) that gets dynamically phosphorylated as RNAP2 progresses through the transcription cycle, therefore regulating each step of transcription from recruitment to termination. Although RNAP2 residue-specific phosphorylation has been characterized in fixed cells by immunoprecipitation-based assays, or in live cells by using tandem gene arrays, these assays can mask heterogeneity and limit temporal and spatial resolution. Our protocol employs multi-colored complementary fluorescent antibody-based (Fab) probes to specifically detect the CTD of the RNAP2 (CTD-RNAP2), and its phosphorylated form at the serine 5 residue (Ser5ph-RNAP2) at a single-copy HIV-1 reporter gene. Together with high-resolution fluorescence microscopy, single-molecule tracking analysis, and rigorous computational modeling, our system allows us to visualize, quantify, and predict endogenous RNAP2 phosphorylation dynamics and mRNA synthesis at a single-copy gene, in living cells, and throughout the transcription cycle. Graphical abstract: Schematic of the steps for visualizing, quantifying, and predicting RNAP2 phosphorylation at a single-copy gene.