Diving deep into the dynamics of the RNA polymerase III transcription machinery

DNA contains the instructions needed for life, but only 1–2% of our genes encode proteins. Many genes instead produce functional RNAs, including transfer RNAs (tRNAs), which are essential for protein synthesis. tRNA genes are transcribed by RNA Polymerase III (RNAPIII), a specialized enzyme recruited through a dynamic interplay between RNAPIII, TFIIIB, TFIIIC, and additional regulatory factors. This thesis investigates how these components cooperate to control tRNA gene transcription and why individual tRNA genes are regulated differently.
Using the budding yeast Saccharomyces cerevisiae as a model system and applying high-throughput approaches such as Epi-Decoder, this thesis identifies new regulators of RNAPIII dynamics. A major discovery is Fpt1, a previously unknown protein that acts as a negative regulator of RNAPIII assembly. Fpt1 binds all tRNA genes, interacts with TFIIIC, and is particularly important under repressive conditions. Further work reveals the structural features required for Fpt1 function and highlights the dual role of TFIIIC as both an activator and repressor of RNAPIII transcription, depending on cellular context.
Finally, this thesis uncovers unexpected flexibility in RNAPIII assembly. TFIIIB can bind independently of TFIIIC at tRNA genes containing upstream TATA-like elements, and some RNAPIII can associate with tRNA genes even in the absence of TFIIIB, suggesting alternative recruitment pathways. Together, these findings reveal that regulation of tRNA transcription is far more dynamic and complex than previously appreciated, while raising new questions about how differential tRNA gene expression is achieved.