ScTFIIIB is composed of three subunits, the TATA-binding protein (TBP), TFIIB-related factor 1 (BRF1) and B double prime 1 (BDP1). Together with TFIIIA, TFIIIC and Pol III, these three components are required and sufficient for reconstituting transcription from all Pol III promoters in Sc.9
ScBRF1 is a paralog of TFIIB and it possesses a structure that is similar to that of TFIIB. It contains an N-terminal zinc ribbon and two direct imperfect repeats, each of which consists of 5 alpha-helices that together adopt a cyclin fold.10
In vertebrate cells, two distinct TFIIIB-activities have been described that both comprise TBP and BDP1, but that differ by either containing TFIIB-related factor 1 (BRF1; component of TFIIIB-β; transcription of type 1 and 2 promoters; ) or TFIIB-related factor 2 (BRF2; component of TFIIIB-α).6,11,12
TFIIIB-α is active in transcription of Pol III genes with gene regulatory elements that are entirely located upstream of the transcription initiation site (type 3 promoter; ) and the BRF2 component of TFIIIB-α has also been cross-linked to genes that contain a combination of gene-internal and gene-external promoter elements.13,14
The evolution of a second TFIIB-related factor at the emergence of vertebrates may have permitted or may have been tolerated by the co-evolution of a novel Pol III promoter type. With respect to the question of whether the evolution of BRF2 led to the co-evolution of a novel promoter type it is noteworthy to mention that promoter elements upstream of the transcription initiation site have also been identified for the U6 and 7SK genes in Drosophila melanogaster
Despite the gene regulatory elements being located upstream of the transcription initiation site, only one isoform of a DmTFIIB-related factor has been described. These results indicate that the evolution of upstream promoters in the Pol III transcription system, at least in insects, may not have been driven by the evolution of a second isoform of a TFIIB-related factor, although we cannot exclude that BRF2 sequences may have been lost in the course of evolution in these species. If ever the appearance of type 3 promoters may not be attributable to the evolution of BRF2 it may be asked whether BRF1 or BRF2 possess other gene- or cell type-specific functions. Today, we only know a single gene in vertebrates (coding for BC200 RNA in humans or the functionally analogous BC1 RNA in rodents) that is transcribed by Pol III and that shows neuronal-specific expression.5
ChIP sequencing and ChIP-on-CHIP experiments demonstrated that the BC200 gene is in physical contact with Pol III.13,16
ChIP sequencing further showed the presence of BDP1 at the BC200 genomic locus. Interestingly, however, neither BRF1, nor BRF2 could be detected at the BC200 gene locus.13,16
This could merely be a technical problem, but it could also indicate that transcription initiation of the BC200 gene may be independent of BRF1 and BRF2. In this case, another, hitherto unidentified protein may replace BRF1/BRF2 for transcription of the BC200 gene in neurons. Taken together, the emergence of BRF1 and BRF2 during evolution led to the appearance of two isoforms of TFIIIB with promoter-specific functions.
TBP was once assumed to be a universal transcription factor.17
This point of view was well understandable at that time, because the discovery that TBP participates in transcription of all three RNA polymerases would not have been anticipated some years earlier and TBP was thought to be generally indispensable for transcription. However since then, several paralogs of TBP have been identified (TRF1; TRF2; TRF3) and their discovery indicated that transcription of certain genes may not depend on TBP.
Although first described as a cell type-specific factor, TRF1 turned later out to be widely expressed in Dm and to replace TBP for transcription by Pol III,18
which was confirmed by ChIP-on-CHIP analyses using affinity-purified anti-TRF1 antibodies and Drosophila genome tiling arrays.19
In addition to its essential functions in Dm Pol III transcription, TRF1 has also been implicated in the transcription of a small subset of Pol II genes.20
No ortholog of TRF1 could hitherto be identified in species other than insects.
Several years later, a second paralog of TBP was identified in mammals, Drosophila melanogaster, Caenorhabditis elegans
and other metazoans.20
TRF2 was shown to be involved in transcription by Pol II,20
but it was demonstrated that recombinant HsTRF2 was inactive in Pol III transcription in vitro.21
The third and latest member of the TBP-family that has been identified is TRF3/TBP2. A gene encoding TRF3 was found in a variety of metazoans, including humans, mice, frogs and fish. Specific functions for TRF3 in differentiation of mouse myoblasts, in zebrafish hematopoiesis and in Xenopus or mouse oocytes have been suggested.20,22
TRF3/TBP2 knockout studies showed that mice had no apparent phenotype except females being sterile due to defective folliculogenesis.23
A possible involvement of TRF3/TBP2 in Pol III transcription has not yet been reported. However, the high degree of conservation between the C-terminal part of human TBP (residues 141–337) and TRF3 (residues 184–374; 92% identity and 95% homology) makes it likely that TRF3 may be functional in Pol III transcription. Moreover, residues in ScTBP that have been reported to be critical for the interaction with ScBRF1 (S261; D263; S282; E284; E286; L287; R299; V306) or with ScBdp1 (H277) have been conserved in both, HsTBP and HsTRF3/TBP2.24
In line with the possibility that TRF3/TBP2 may be able to replace TBP in Pol III transcription, it has been suggested that TRF3/TBP2 may be a TBP replacement factor in cells that contain low levels of TBP.22