Stepwise Recruitment of Components of the Preinitiation Complex by Upstream Activators In Vivo

ABSTRACT

Recently, it was found that if either the TATA binding protein or RNA polymerase II holoenzyme is artificially tethered to a promoter, transcription is activated. This finding provided presumptive evidence that upstream activating proteins function by recruiting components of the preinitiation complex (PIC) to the promoter. To date, however, there have been no studies demonstrating that upstream factors actually recruit components of the PIC to the promoter in vivo. Therefore, we have studied the mechanism of action of two disparate transactivating domains. We present a series of in vivo functional assays that demonstrate that each of these proteins targets different components of the PIC for recruitment. We show that, by targeting different components of the PIC for recruitment, these activating domains can cooperate with each other to activate transcription synergistically and that, even within one protein, two different activating subdomains can activate transcription synergistically by cooperating to recruit different components of the PIC. Finally, considering our work together with previous studies, we propose that certain transcription factors both recruit components of the PIC and facilitate steps in transcriptional activation that occur subsequent to recruitment.

ACKNOWLEDGMENTS

We thank J. T. Lis for critical review of the manuscript. We thank A. Berk for pGal4-VP16Δ456, W. Schaffner for pGal4-Sp1, W. Herr for pCGNTBP and pCGNTBP_AS, M. R. Green for pG₂E1B-CAT and pE1B-CAT, R. G. Roeder for p6His-T-hTBP, I. Sadowski for PM1, and P. Broad for pBXL₁and pL6EC.

This work was supported by a grant from the NIH.