Yick Hin Ling, Chloe Liang, Sixiang Wang, Carl Wu
Eukaryotic gene expression is orchestrated by RNA polymerases (RNAPI, II, and III) and associated factors, yet their real-time dynamics remain obscure. Using single-molecule tracking in living yeast, we quantified the kinetics of 58 proteins encompassing three RNAP machineries. RNAPI and RNAPIII preinitiation complexes (PICs) engage in long-lived chromatin interactions, contrasting with transient RNAPII PICs. We further report kinetics of RNAPII-associated factors for elongation, histone modification, C-terminal domain (CTD) modification, RNA processing, and termination. Many elongation factors show brief rather than persistent association, suggesting dynamic interactions with factor exchange and allowing a potential repertoire of regulatory events. CTD truncation reduces U1 small nuclear ribonucleoprotein residence time and intron retention in ribosomal protein genes, providing insights into cotranscriptional splicing. Our findings establish a framework of dynamic interactions of RNAP machineries.