Abstract
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 hTFIIIC1-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 anti-HsBdp1 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 approximately 90-150 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.
Highlights
RNA polymerase III transcribes genes encoding small, untranslated RNAs including tRNA, 5 S rRNA, and U6 small nuclear RNA genes
In an attempt to identify the proteins required for the reconstitution of TFIIIC1 activity, we established a purification scheme (Fig. 1A) that allows the preparation of highly purified protein samples with TFIIIC1 activity
TFIIIC1 activity was split into two distinct peaks upon chromatography over MonoS (Fig. 1B)
Summary
RNA polymerase III transcribes genes encoding small, untranslated RNAs including tRNA, 5 S rRNA, and U6 small nuclear RNA genes (reviewed in Ref. 1). We demonstrate that a 250kDa protein, recognized in Western blots by anti-HsBdp[1] antibodies, co-eluted with TFIIIC1 activity over several purification steps.
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