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 pre-initiation complexes (PICs) engage in long-lived chromatin interactions, contrasting with transient RNAPII PIC. 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, allowing a potential repertoire of regulatory events. CTD truncation reduces U1 snRNP residence time and intron retention in ribosomal protein genes, providing insights into co-transcriptional splicing. Our findings establish a framework of dynamic interactions of RNAP machineries.
Ling et al. (2026) studied this question.