Effects of sumoylation-impairing mutation of TFIIF subunit Tfg1 on genome-wide RNA polymerase II (RNAPII) levels in budding yeast

Many transcription-related proteins, including components of the RNA polymerase II (RNAPII) general transcription factors (GTFs), have been shown through proteomics studies to be modifiable by SUMO conjugation. However, we determined that the large subunit of TFIIF, Tfg1, is likely the major target of sumoylation among GTFs in budding yeast. The primary site of sumoylation on Tfg1 is Lys residues 60 or 61, which lies in a region of the protein that is proximal to the Rpb2 subunit of RNAPII when the GTF-RNAPII preinitiation complex (PIC) assembles at promoters. As TFIIF and RNAPII are highly functionally and physically linked, we examined whether blocking Tfg1 sumoylation through Lys-to-Arg mutations could affect the recruitment or association of RNAPII with chromatin. RNAPII ChIP-seq was performed in strains expressing wild-type (Tfg1-HA) or sumoylation-deficient (Tfg1-K60,61R-HA) forms of Tfg1. Indeed, nearly half of all protein-coding genes showed significantly reduced RNAPII levels in the mutant strain. These data suggest that sumoylation of TFIIF plays an important role in regulating the dynamics of RNAPII association with genes. Two independent replicates of RNAPII ChIP-seq from Tfg1-HA and Tfg1-K60,61R-HA strains.