Live-cell single-molecule dynamics of eukaryotic RNA polymerase machineries

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Eukaryotic gene expression relies on RNA polymerases (RNAPI, II, III) and associated factors.
• The real-time dynamics of these protein interactions within living cells are not well understood.

Purpose of the Study:

• To quantify the kinetics of proteins involved in the three main RNA polymerase machineries in yeast.
• To elucidate the dynamic interactions of RNA Polymerase II-associated factors during transcription.

Main Methods:

• Single-molecule tracking in living yeast cells.
• Quantification of protein kinetics for 58 proteins across RNAPI, RNAPII, and RNAPIII machineries.

Main Results:

• RNAPI and RNAPIII pre-initiation complexes (PICs) exhibit long-lived chromatin interactions, unlike transient RNAPII PICs.
• Many RNAPII elongation factors display brief associations, indicating dynamic exchange and regulatory potential.
• CTD truncation affects U1 snRNP residence time and intron retention in ribosomal protein genes, impacting co-transcriptional splicing.

Conclusions:

• Established a framework for understanding the dynamic interactions of RNA polymerase machineries in real-time.
• Provided insights into the regulation of transcription, RNA processing, and splicing through dynamic protein-chromatin interactions.