biostudies-literaturevan Breugel MEEuropean Research CouncilDutch Research Council (NWO)NIGMS NIH HHS4205-4221.e9https://www.ebi.ac.uk/biostudies/studies/S-EPMC11289708biostudies-literatureUnknown83(23)Transcription of tRNA genes by RNA polymerase III (RNAPIII) is tuned by signaling cascades. The emerging notion of differential tRNA gene regulation implies the existence of additional regulatory mechanisms. However, tRNA gene-specific regulators have not been described. Decoding the local chromatin proteome of a native tRNA gene in yeast revealed reprogramming of the RNAPIII transcription machinery upon nutrient perturbation. Among the dynamic proteins, we identified Fpt1, a protein of unknown function that uniquely occupied RNAPIII-regulated genes. Fpt1 binding at tRNA genes correlated with the efficiency of RNAPIII eviction upon nutrient perturbation and required the transcription factors TFIIIB and TFIIIC but not RNAPIII. In the absence of Fpt1, eviction of RNAPIII was reduced, and theMolecular cellLocus-specific proteome decoding reveals Fpt1 as a chromatin-associated negative regulator of RNA polymerase III assembly.PMC11289708755695R35 GM145217731.015.405016.Veni.192.071Mittal CDel Cortona AHoekman Lvan Leeuwen FPugh BFKluin RJCLieftink CMenezes RXMalik Mvan Breugel MEvan Kruijsbergen Ivan den Brand TBeijersbergen RLLenstra TLVerstrepen KJBrouwer Ivan Welsem TfalseLocus-specific proteome decoding reveals Fpt1 as a chromatin-associated negative regulator of RNA polymerase III assembly.Transcription of tRNA genes by RNA polymerase III (RNAPIII) is tuned by signaling cascades. The emerging notion of differential tRNA gene regulation implies the existence of additional regulatory mechanisms. However, tRNA gene-specific regulators have not been described. Decoding the local chromatin proteome of a native tRNA gene in yeast revealed reprogramming of the RNAPIII transcription machinery upon nutrient perturbation. Among the dynamic proteins, we identified Fpt1, a protein of unknown function that uniquely occupied RNAPIII-regulated genes. Fpt1 binding at tRNA genes correlated with the efficiency of RNAPIII eviction upon nutrient perturbation and required the transcription factors TFIIIB and TFIIIC but not RNAPIII. In the absence of Fpt1, eviction of RNAPIII was reduced, and the2023-01-01T00:00:00Z2023 Dec2025-04-04T01:24:13.836Z2025-04-04T01:24:13.836ZS-EPMC112897083799569110.1016/j.molcel.2023.10.037