Absolute gene occupancies by RNA polymerase III, TFIIIB, and TFIIIC in Saccharomyces cerevisiae.

ABSTRACT: A major limitation of chromatin immunoprecipitation lies in the challenge of measuring the immunoprecipitation effectiveness of different proteins and antibodies and the resultant inability to compare the occupancies of different DNA-binding proteins. Here we present the implementation of a quantitative chromatin immunoprecipitation assay in the RNA polymerase III (pol III) system that allowed us to measure the absolute in vivo occupancy of pol III and its two transcription factors, TFIIIC and TFIIIB, on a subset of pol III genes. The crucial point of our analysis was devising a method that allows the accurate determination of the immunoprecipitation efficiency for each protein. We achieved this by spiking every immunoprecipitation reaction with the formaldehyde cross-linked in vitro count

SUBMITTER: Soragni E 

PROVIDER: S-EPMC2546553 | biostudies-literature | 2008 Sep

REPOSITORIES: biostudies-literature