Yeast nuclear extract contains two major forms of RNA polymerase II mediator complexes

The yeast Mediator complex is required for transcription by RNA polymerase II (pol II) in vivo and in vitro. This complex of over 20 polypeptides asso ciates with pol II and is recruited to transcription complexes at promoters . Previous isolations of yeast Mediator-containing complexes in different l aboratories have identified several distinct complexes. To identify the maj or forms of Mediator in yeast, Mediator was isolated from nuclear extracts using a two-step chromatographic procedure, avoiding ion exchange chromatog raphy and high salt conditions to prevent dissociation of subunits during p urification. Components of the Mediator complexes were identified by mass s pectrometry and Western analysis. The major form of Mediator, termed pol II Med, contained pol II and Mediator, including the Srb8-11 module. A second lower molecular size complex was also identified, termed Mediator core (Me dc), which lacked pol II, Srb8-11, Rox3, Nut1, and the Rgr1 module. Both of these complexes were active in transcription in vitro, although the Medc c omplex had significantly lower activity and could compete with the activity of the pol II Med complex in vitro.