Human Mediator Enhances Activator-Facilitated Recruitment of RNA Polymerase II and Promoter Recognition by TATA-Binding Protein (TBP) Independently of TBP-Associated Factors

Abstract

Mediator is a general cofactor implicated in the functions of many transcriptional activators. Although Mediator with different protein compositions has been isolated, it remains unclear how Mediator facilitates activator-dependent transcription, independent of its general stimulation of basal transcription. To define the mechanisms of Mediator function, we isolated two forms of human Mediator complexes (Mediator-P.5 and Mediator-P.85) and demonstrated that Mediator-P.5 clearly functions by enhancing activator-mediated recruitment of RNA polymerase II (pol II), whereas Mediator-P.85 works mainly by stimulating overall basal transcription. The coactivator function of Mediator-P.5 was not impaired when TATA-binding protein (TBP) was used in place of TFIID, but it was abolished when another general cofactor, PC4, was omitted from the reaction or when Mediator-P.5 was added after pol II entry into the preinitiation complex. Moreover, Mediator- P.5 is able to enhance TBP binding to the TATA box in an activator-dependent manner. Our data provides biochemical evidence that Mediator functions by facilitating activator-mediated recruitment of pol II and also promoter recognition by TBP, both of which can occur in the absence of TBP-associated factors in TFIID.

ACKNOWLEDGMENTS

We thank R. D. Kornberg for human Med7 cDNA; D. Reinberg for pG₅MLT and pΔMLP DNA templates; R. G. Roeder for antibodies against TRAP240, TRAP230, TRAP220, TRAP170, TRAP150, TRAP100, and TRAP80; and R. Tjian for anti-ARC105, anti-CRSP150, and anti-CRSP130 antibodies. We are also grateful to D. Luse for advice on immobilized DNA template and P. de Haseth, D. Luse, E. Stavnezer, and D. Samols for comments on the manuscript.

C.-M.C. is a Mt. Sinai Health Care Foundation Scholar. This work was supported by grants GM59643 and CA81017 from the National Institutes of Health.