An RNA Polymerase II Complex Containing All Essential Initiation Factors Binds to the Activation Domain of PAR Leucine Zipper Transcription Factor Thyroid Embryonic Factor

Abstract

Transcription initiation of protein-encoding genes involves the assembly of RNA polymerase II and a number of general transcription factors at the promoter. A mammalian RNA polymerase II complex containing all of the components required for promoter-specific transcription initiation can be isolated by immunopurification with a monoclonal antibody directed against the cyclin-dependent kinase CDK7, a subunit of the general transcription factor TFIIH. In vitro transcription by this immunopurified RNA polymerase II complex is effectively stimulated by thyroid embryonic factor (TEF), a basic leucine zipper transcription factor. Thus, the RNA polymerase II complex must also contain components required for activated transcription that interact with the transactivation domain of TEF. This conjecture was verified by affinity selection experiments in which the TEF transcription activation domain was used as a bait. Indeed, an RNA polymerase II complex containing all of the accessory proteins required for transcription initiation can be enriched by its affinity to recombinant proteins containing the TEF transactivation domain. These results are compatible with a mechanism by which TEF can recruit an RNA polymerase II holoenzyme to the promoter in a single step.

ACKNOWLEDGMENTS

We are grateful to Rick Young for the antiserum against human SRB7, Erich Nigg for the CDK7 monoclonal antibody, Moshe Yaniv for hBrm antibodies, and Shelley Berger for hGCN5 antibodies. We thank Nicolas Roggli for expert preparation of the figures, and Steve Brown, Juergen Ripperger, David Gdula, Philippe Georgel, and Raphael Sandaltzopoulos for their critical reading of the manuscript.

This work was supported by the Canton of Geneva, the Swiss National Science Foundation, and a postdoctoral fellowship to V.O. from the Roche Research Foundation, Basel, Switzerland.