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It has been shown that under specific conditions, transcription of protein coding genes can be efficiently initiated by RNA polymerase (pol) III in vitro. We examined the formation and composition of such pol III transcription complexes on the duck histone H5 and alphaA-globin promoters and found that the essential step for the formation of pol III transcription complexes on these pol II promoters was the stable binding of transcription factor (TF) IIIB-beta. For this process, the intact TFIIIB-beta complex, consisting of TBP and associated factors (TAFs) was needed and the prior association of pol III assembly factors was not necessary. We demonstrate for the first time that hTFIIIB-beta alone is able to bind to pol II promoter DNA. This resulted in a very stable complex which was resistant to high concentrations of heparin. Although immunodepletion revealed that TBP is essentially required for complex formation, other components of hTFIIIB-beta must also be involved, since TBP itself is unable to form heparin-resistant complexes and does not mediate pol III commitment per se. pol III is recruited to these pol II promoters in a strictly TFIIIC1 dependent way. After binding of TFIIIB-beta, the addition of TFIIIC1 and pol III were sufficient to yield productive pol III transcription complexes, which utilized the correct pol II initiation site. From these findings, we postulate that TFIIIC1 is involved in the recruitment of pol III and may thus form a bridge between TFIIIB-beta and the enzyme. This finding provides the first evidence for functional contacts between TFIIIC1 and pol III, which could be of general importance for the assembly of pol III transcription complexes.

We analysed the formation of transcription complexes on the H5 gene of the duck which is efficiently transcribed in HeLa cell extracts in vitro. Upon deletion of its TATA-box, the fidelity of transcription of the H5 gene is maintained, although the efficiency of this process is significantly reduced. Selective inactivation of TFIID in whole cell extracts and reconstitution experiments either with human recombinant TFIID or a protein fraction from duck erythrocytes enriched in TFIID show that transcription of the TATA-less H5 promoter nevertheless requires the protein TFIID. Screening of promoter elements which could indirectly mediate the interaction of TFIID with a TATA-less H5 promoter led to the identification of a sequence element located about 40 base-pairs downstream from the H5 initiation site that shows partial homology to the USF consensus sequence. In electrophoretic mobility shift and footprinting studies we demonstrated a specific interaction of the erythroid factor USF (eUSF) with this downstream element. By isolating active transcription complexes we found that all components required for correct initiation remain stably associated with the H5 promoter irrespective of the presence or absence of the TATA box. Moreover, the reconstitution of eUSF and TFIID-depleted transcription complexes with purified protein fractions demonstrate that not only TFIID but also eUSF essentially participates in complex formation even on H5 promoter mutations lacking the TATA-box. Mutual interactions between eUSF and TFIID appear to stabilize the binding of TFIID in the presence or absence of its proper binding site.

The Xenopus tropicalis U6 gene is very poorly transcribed both when introduced into human cells by transfection, and in human cell-free extracts. By analysis of hybrid promoters constructed from human and Xenopus sequences in various combinations, we show that species specificity is mediated by the proximal sequence elements (PSEs) of the promoters. We demonstrate the PSE-dependence of U6 transcription in a fractionated extract of HeLa cells. One of the fractions required for transcription contains an activity designated PSE-binding protein (PBP), previously shown to bind to the PSE of the mouse U6 gene. Binding of PBP to various wild-type and hybrid U6 PSE sequences correlates with their activity in transcription in HeLa cell extracts. This provides strong evidence that PBP is the PSE-binding factor involved in U6 transcription. In addition, it suggests that the differential affinities of the promoters for PBP is responsible for the observed species specificity. The divergence between U snRNA promoters in different species contrasts with the relatively strong conservation of other families of RNA polymerase II and III transcribed gene promoters. Possible mechanisms by which this diversity could be generated are discussed.