A spectrum of mechanisms for the assembly of the RNA polymerase II transcription preinitiation complex.

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.

Structure of the genes encoding transcription factor IIB and TATA box-binding protein from Drosophila melanogaster.

To investigate the structure and regulation of the genes encoding components of the basal RNA polymerase II transcriptional machinery, the Drosophila melanogaster genes encoding transcription factor IIB (TFIIB) and the TATA-box-binding protein (TBP) were isolated and characterized. In the TBP gene, a single intron bisects the sequences that encode the two repeated DNA-binding domains of TBP, which supports the notion that TBP evolved from an earlier form that possessed only a single domain. The transcription start points (tsp) were determined, and there was a good correlation between the tsp that were used in vivo and transcription reactions in vitro. The TFIIB and TBP genes have several similar features, which include high A + T content (68 to 74%) upstream from the tsp, multiple copies of an ATTATTATT sequence motif in the proximal promoter region, the absence of a consensus TATA-box element, and small introns (55 to 64 nucleotides). These genes should be useful in the combined genetic and biochemical analysis of TFIIB and TBP in D. melanogaster.

Identification of a minimal set of proteins that is sufficient for accurate initiation of transcription by RNA polymerase II.

In eukaryotes, initiation of mRNA synthesis is a multistep process that is carried out by RNA polymerase II and auxiliary factors that are commonly referred to as basal or general factors. In this study accurate initiation of transcription was reconstituted with purified, Escherichia coli-synthesized TFIIB, TBP (the TATA box-binding polypeptide of the TFIID complex), and the 30-kD subunit of TFIIF (also known as RAP30), along with purified, native RNA polymerase II from Drosophila embryos, calf thymus, or HeLa cells. This minimal set of factors was able to transcribe a subset of the promoters tested. The addition of both subunits of TFIIE and the 74-kD subunit of TFIIF increased the efficiency of transcription by a factor of 2 to 4. In contrast, the inclusion of a crude TFIID fraction from Drosophila embryos in place of recombinant TBP resulted in a strong dependence on TFIIE. By gel mobility-shift analysis, TFIIB, TBP, RAP30, and polymerase were able to assemble into DB and DBPolF30 complexes with transcriptionally competent (wild type or initiator mutant), but not with transcriptionally inactive (TATA and TATA/initiator mutant), versions of the Drosophila Adh promoter. Thus, it appears that RNA polymerase II is able to initiate transcription subsequent to assembly of the DBPolF30 complex, which is a minitranscription complex that represents the central core of the RNA polymerase II transcriptional machinery.