biostudies-literatureVersluis PUS Government National Institutes of HealthNIGMS NIH HHSNew York State Stem Cell ScienceNIH HHSNational Science Foundation2856-2869.e9https://www.ebi.ac.uk/biostudies/studies/S-EPMC11486293biostudies-literatureUnknown84(15)RNA polymerase II (RNA Pol II)-mediated transcription is a critical, highly regulated process aided by protein complexes at distinct steps. Here, to investigate RNA Pol II and transcription-factor-binding and dissociation dynamics, we generated endogenous photoactivatable-GFP (PA-GFP) and HaloTag knockins using CRISPR-Cas9, allowing us to track a population of molecules at the induced Hsp70 loci in Drosophila melanogaster polytene chromosomes. We found that early in the heat-shock response, little RNA Pol II and DRB sensitivity-inducing factor (DSIF) are reused for iterative rounds of transcription. Surprisingly, although PAF1 and Spt6 are found throughout the gene body by chromatin immunoprecipitation (ChIP) assays, they show markedly different binding behaviors. Additionally, we found that PAF1 and Spt6 are only recruited after positive transcription elongation factor (P-TEFb)-mediated phosphorylation and RNA Pol II promoter-proximal pause escape. Finally, we observed that PAF1 may be expendable for transcription of highly expressed genes where nucleosome density is low. Thus, our live-cell imaging data provide key constraints to mechanistic models of transcription regulation.Molecular cellLive-cell imaging of RNA Pol II and elongation factors distinguishes competing mechanisms of transcription regulation.PMC11486293RM1 GM139738C029155S10OD0185162139899S10 OD018516LSM880Ebenezer JGraham TGWVersluis PDarzacq XLis JTEng VZipfel WRfalseLive-cell imaging of RNA Pol II and elongation factors distinguishes competing mechanisms of transcription regulation.RNA polymerase II (RNA Pol II)-mediated transcription is a critical, highly regulated process aided by protein complexes at distinct steps. Here, to investigate RNA Pol II and transcription-factor-binding and dissociation dynamics, we generated endogenous photoactivatable-GFP (PA-GFP) and HaloTag knockins using CRISPR-Cas9, allowing us to track a population of molecules at the induced Hsp70 loci in Drosophila melanogaster polytene chromosomes. We found that early in the heat-shock response, little RNA Pol II and DRB sensitivity-inducing factor (DSIF) are reused for iterative rounds of transcription. Surprisingly, although PAF1 and Spt6 are found throughout the gene body by chromatin immunoprecipitation (ChIP) assays, they show markedly different binding behaviors. Additionally, we found that PAF1 and Spt6 are only recruited after positive transcription elongation factor (P-TEFb)-mediated phosphorylation and RNA Pol II promoter-proximal pause escape. Finally, we observed that PAF1 may be expendable for transcription of highly expressed genes where nucleosome density is low. Thus, our live-cell imaging data provide key constraints to mechanistic models of transcription regulation.2024-01-01T00:00:00Z2024 Aug2026-04-13T13:03:53.197Z2026-04-07T13:29:16.769ZS-EPMC114862933912184310.1016/j.molcel.2024.07.009