A Spectrum of Mechanisms for the Assembly of the RNA Polymerase II Transcription Preinitiation Complex

Abstract

To explore the diversity in the mechanisms of basal transcription by RNA polymerase II, we have employed a novel biochemical approach that involves perturbation of the transcription reaction with exogenously added TFIIB or TATA box-binding protein (TBP). Under these conditions, we observe promoter-selective inhibition of transcription by excess TFIIB or excess TBP. This inhibition occurs at the level of basal transcription, because it is observed with minimal promoters that comprise only the TATA box and initiation site sequences as well as with preparations of basal transcription factors that have been purified to greater than 90% homogeneity. In addition, the excess basal factors inhibit the assembly of a functional preinitiation complex but do not inhibit transcription initiation from preassembled preinitiation complexes. A study of several promoters revealed a reciprocal trend in the promoter specificity of inhibition by excess TFIIB versus that by excess TBP. At opposite ends of this spectrum, promoters are strongly inhibited by excess TFIIB but not excess TBP and vice versa. These results reveal the existence of a spectrum of mechanisms for preinitiation complex assembly at different promoters. The mechanistic preference appears to be specified by the aggregate of basal promoter elements rather than by an individual component, such as the TATA box or initiation site sequence. This spectrum provides a new parameter by which differences in the function of minimal class II promoters can be analyzed in the context of both basal and regulated transcription.