Interplay of Positive and Negative Regulators in Transcription Initiation by RNA Polymerase II Holoenzyme

ABSTRACT

Activation of protein-encoding genes involves recruitment of an RNA polymerase II holoenzyme to promoters. Since the Srb4 subunit of the holoenzyme is essential for expression of most class II genes and is a target of at least one transcriptional activator, we reasoned that suppressors of a temperature-sensitive mutation in Srb4 would identify other factors generally involved in regulation of gene expression. We report here that MED6 and SRB6, both of which encode essential components of the holoenzyme, are among the dominant suppressors and that the products of these genes interact physically with Srb4. The recessive suppressors include NCB1 (BUR6),NCB2, NOT1, NOT3, NOT5, and CAF1, which encode subunits of NC2 and the Not complex. NC2 and Not proteins are general negative regulators which interact with TATA box binding protein (TBP). Taken together, these results suggest that transcription initiation involves a dynamic balance between activation mediated by specific components of the holoenzyme and repression by multiple TBP-associated regulators.

ACKNOWLEDGMENTS

We thank P. Sharp, M. Green, V. Myer, and H. Madhani for advice and discussions. We thank F. Holstege, M. Collart, M. Green, Y.-J. Kim, J. Madison, J. Reese, K. Struhl, C. Wilson, and F. Winston for kind gifts of extracts, strains, plasmids, and antibodies. We thank A. S. Lee for technical assistance.

J.J.W. is a predoctoral fellow of the National Science Foundation. E.G.J. is a predoctoral fellow of the Howard Hughes Medical Institute. This work was supported by National Institutes of Health grants to R.A.Y.