Identification of proteins associated with RNA polymerase III using a modified tandem chromatin affinity purification

•A modified TChAP protocol after in vivo cross-link is precisely described.•TChAP procedure identifies more associated proteins than traditional TAP-MS.•Purified TFIIIA is not associated either with Pol III machinery or 5S rDNA genes.•We capture free Pol III and Pol III engaged in transcription reinitiation.

To identify the proteins associated with the RNA polymerase III (Pol III) machinery in exponentially growing yeast cells, we developed our own tandem chromatin affinity purification procedure (TChAP) after in vivo cross-link, allowing a reproducible and good recovery of the protein bait and its associated partners. In contrast to TFIIIA that could only be purified as a free protein, this protocol allows us to capture free Pol III together with Pol III bound on its target genes. Transcription factors, elongation factors, RNA‐associated proteins and proteins involved in Pol III biogenesis were identified by mass spectrometry. Interestingly, the presence of all the TFIIIB subunits found associated with Pol III together with the absence of TFIIIC and chromatin factors including histones suggest that DNA‐bound Pol III purified using TChAP is mainly engaged in transcription reinitiation.