Transcription of class III genes is conducted by multi-protein complexes consisting of polymerase III itself and several transcription factors. We established a reconstituted in vitro transcription system from which the autoantigen La was removed by immunodepletion. This system showed no RNP formation, but was still fully active in transcription. Supplementing such La-free transcription reactions with recombinant La restored the formation of La complexes with the newly synthesised RNA, but did not lead to enhanced transcription efficiency. Furthermore, we developed a technique for the generation and isolation of transcription complexes, assembled from purified transcription factors and isolated by glycerol centrifugation. These complexes were fully competent to re-initiate RNA synthesis but they were not associated with La and their transcription rate could not be stimulated by addition of recombinant La. Therefore, we conclude that La does not act as a human polymerase III transcription factor.

The general human RNA polymerase III transcription factor (TF) IIIC1 has hitherto been ill defined with respect to the polypeptides required for reconstitution of its activity. Here we identify Homo sapiens TFIIIB؆ (HsBdp1) as an essential component of hTFIIIC1 and hT-FIIIC1-like activities. Several forms of HsBdp1 are described. The 250-kDa form of HsBdp1, also designated the "transcription factor-like nuclear regulator," strictly co-eluted with TFIIIC1 activity over multiple chromatographic purification steps as revealed by Western blot with anti-HsBdp1 antibodies and by MALDI-TOF analysis. In addition, TFIIIC1 activity could be depleted from partially purified fractions with antiHsBdp1 antibodies but not with control antibodies. Moreover, highly purified recombinant HsBdp1 could replace TFIIIC1 activity in reconstituted transcription of the VAI gene in vitro. Furthermore, smaller proteins of ϳ90 -150 kDa that were recognized by anti-HsBdp1 antibodies co-eluted with TFIIIC1-like activity. Finally, cytoplasmic extracts from differentiated mouse F9 fibroblast cells that lacked TFIIIC1 activity could be made competent for transcription of the VA1 gene by the addition of TFIIIC1, TFIIIC1-like, or recombinant HsBdp1. These results suggest that HsBdp1 proteins represent essential components of TFIIIC1 and TFIIIC1-like activities.RNA polymerase III transcribes genes encoding small, untranslated RNAs including tRNA, 5 S rRNA, and U6 small nuclear RNA genes (reviewed in Ref. 1). In the yeast Saccharomyces cerevisiae, genes transcribed by RNA polymerase III are governed by promoter elements comprised of A and C boxes (type 1) or A and B boxes (type 2) that are located downstream of the transcription initiation site. Two transcription factors (TF), 1 TFIIIB and TFIIIC, are necessary and sufficient for the transcription of type 2 genes (tRNA), whereas transcription of the type 1 5 S rRNA gene requires, in addition, TFIIIA. S. cerevisiae (Sc) TFIIIC is a stable complex of six polypeptides, whereas ScTFIIIB consists of a less stable association of three components, namely the ScTBP, ScBrf1, and ScBdp1 proteins (reviewed in Refs.

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By employing purified transcription factors and RNA polymerase III (pol III), we generated active pol III transcription complexes on the human 5S rRNA gene. These large complexes were separated by size exclusion chromatography from non- incorporated proteins. In addition, we succeeded in isolating specific intermediate stages of complex formation. Such isolated partial complexes require complementation with the missing activities for full transcription activity. One central finding is that a 5S DNA-TFIIIA-TFIIIC2-TFIIIBbeta complex could be isolated which had been assembled in the absence of the general pol III transcription factor IIIC1. Thus TFIIIC1 is not an assembly factor for other transcription factors. Although pol III has the potential to bind unspecifically to DNA, such polymerase molecules cannot be rendered initiation competent by direct recruitment to a 5S DNA-TFIIIA-TFIIIC2- TFIIIBbeta complex, but this process strictly requires additional TFIIIC1 activity. This clearly demonstrates that in contrast to yeast cells, hTFIIIB(beta), although required, does not suffice for the functional recruitment of polymerase III. These data document that TFIIIC1 is the second transcription factor required for the recruitment of pol III in mammalian cells.

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