RNA polymerase II (Pol II) is a 12 subunit protein complex from yeast to human that is required for gene expression. Gdown1 containing Pol II [Pol II (G)] is a special form of Pol II that is catalytically inactive and heavily depends on the 30-subunit Mediator complex for its activator and basal dependent function in vitro. Here we report for the first time, the identification and the generation of a 15-subunit human Mediator complex via the novel multibac baculovirus expression system that is fully responsive to Pol II (G). Our results show complete recovery of Pol II (G) dependent transcription both with full 30-subunit Mediator and also with 15-subunit recombinant Mediator that we synthesized. Moreover, we also show that the recombinant Mediator interacts with Pol II (G) as well. These results enlighten us towards understanding how a certain population of Pol II that is involved in selected gene regulation is activated by Mediator complex. | Turkish Journal of Biology Turk J Biol 2021 45 253-261 http biology TÜBİTAK Research Article doi biy-2009-12 Reconstitution of Pol II G responsive form of the human Mediator complex 1 2 Murat Alper CEVHER 1 Department of Molecular Biology and Genetics Faculty of Science Bilkent University Ankara Turkey 2 Visiting Assistant Professor Laboratory of Biochemistry and Molecular Biology The Rockefeller University New York USA Received Accepted Published Online Final Version Abstract RNA polymerase II Pol II is a 12 subunit protein complex from yeast to human that is required for gene expression. Gdown1 containing Pol II Pol II G is a special form of Pol II that is catalytically inactive and heavily depends on the 30-subunit Mediator complex for its activator and basal dependent function in vitro. Here we report for the first time the identification and the generation of a 15-subunit human Mediator complex via the novel multibac baculovirus expression system that is fully responsive to Pol II G . Our results show complete recovery of Pol II G dependent transcription both with full 30-subunit Mediator and also with 15-subunit recombinant Mediator that we synthesized. Moreover we also show that the recombinant Mediator interacts with Pol II G as well. These results enlighten us towards understanding how a certain population of Pol II that is involved in selected gene regulation is activated by Mediator complex. Key words Mediator Pol II G Gdown1 transcription reconstitution multibac 1. Introduction dynamically to the core Mediator Blazek et al. 2005 . The Mediator complex is a multisubunit coactivator complex Mediator complex primarily transmits signals from general consisting of different modules and is the chief regulator of transcription factors and activators to Pol II regulating the transcription by RNA polymerase II Pol II Allen and the function of the enzyme in the preinitiation initiation Taatjes 2015
