Bioinformatic analysis of the recently completed Trypanosoma brucei
genome suggests a scarcity of easily recognizable basal transcription factors and a limited repertoire of putative regulatory proteins (5
). Accordingly, the challenge is to analyze, genetically and biochemically, the function of candidate proteins that contain weak homology to known factors. We identified and characterized a candidate trypanosome TFIIB, which upon experimental evaluation demonstrated many of the key features of known TFIIB proteins. Specifically, our designated tTFIIB is similar in size (38 kDa) to well-studied TFIIBs, contains the metal binding residues found in the amino-terminal zinc ribbon domain of these TFIIBs, and has one of the two carboxyl-terminal core domain repeats common to TFIIBs. We find that trypanosomal TFIIB is essential for cell growth, is required for SL RNA gene transcription, and interacts with both tTBP and RNA polymerase II.
TFIIB is essential in Saccharomyces cerevisiae
and mammals as it functions as a key basal transcription factor at both mRNA and snRNA polymerase II-dependent promoters (14
). We compare and contrast the two promoter types below and discuss the features of tTFIIB germane to each.
Many mRNA promoters contain a promoter-proximal TATA element. During preinitiation complex formation, TBP binds to this element, bends DNA, and recruits TFIIB. Direct interaction of TFIIB with sequences flanking the TATA box (specifically, the TFIIB recognition element, BRE) helps stabilize the TBP-promoter-TFIIB interaction (23
). This ternary complex recruits the TFIIF-RNA polymerase II complex and dictates transcriptional direction. Mutational studies of yeast highlight the role of TFIIB (SUA7
) in transcriptional start site selection (30
Promoter architecture and protein requirements at snRNA start sites differ from those of mRNA promoters (16
). The human U1 snRNA promoter resembles the trypanosome SL RNA promoter as they both recruit RNA polymerase II and contain a proximal sequence element (PSE for human U1 snRNA and PBP-1E for trypanosome SL RNA) centered 50 to 70 bp upstream of the transcriptional start site. In human cells, while there is neither a TATA box nor a BRE, TBP is essential for U1 snRNA production. TBP interacts with SNAPc, a five-polypeptide complex uniquely associated with snRNA promoters. Finally, in vitro transcription studies reveal TFIIB to be a necessary member of the snRNA transcriptional machinery (22
). Overall, while it is not known whether the precise roles of TFIIB are comparable at mRNA and snRNA promoters, in both cases, it is thought that TFIIB bridges the interaction of RNA polymerase II with the TBP-DNA complex (16
Our trypanosome TFIIB studies were carried out at the small nuclear SL RNA gene promoter, as this is the only identified RNA polymerase II promoter in these parasites. Beyond requiring RNA polymerase II, several similarities exist between human U1 snRNA and trypanosome SL RNA synthesis. In trypanosomes, a slimmed-down SNAPc, containing homologs to two of the five human SNAPc proteins, associates with the SL RNA gene promoter. Depletion studies of nuclear extract reveal that this protein complex is essential (9
). tTBP also plays a central role in SL RNA production as it is required for transcription (9
). Additionally, as in HeLa nuclear extracts, tTBP is found to interact with tSNAPc. Finally, as demonstrated by our depletion and reconstitution studies as well as by our RNAi studies, tTFIIB is requisite for transcriptional activity. In summary, the emerging picture of trypanosomatid transcription at the SL RNA gene promoter is somewhat reminiscent of that found at snRNA promoters in higher eukaryotes.
To compare our results in the framework of TFIIB function at mRNA promoters is difficult, given that there are neither defined mRNA promoters nor obvious TATA boxes or BREs in trypanosomes. A further complication is the notion that transcription initiation may be a random event in trypanosomes (8
). A discussion of how TbTFIIB deviates from higher eukaryotic homologs in manners applicable to mRNA synthesis is outlined below.
First, mutational and crystallographic studies reveal two residues in human TFIIB (Val283
) that are necessary for the specific binding of TFIIB to the BRE, flanking the TATA element in core promoters (23
). These residues are not conserved in TbTFIIB (the equivalent residues are proline and glutamine, respectively) and fall within the missing second repeat of the carboxyl-terminal core domain. The absence of TATA boxes and BREs near potential pre-mRNA start sites is consistent with this sequence variation (27
Second, mutational analysis of yeast TFIIB demonstrates that it selects the transcriptional start site of pre-mRNAs (30
). Curiously, as shown in Fig. , many of the residues critical for positioning polymerase are not conserved in tTFIIB. This lack of conservation may be interpreted to mean that (i) tTFIIB is relieved of this role, particularly given the predicted random nature of transcription initiation for protein-coding genes in trypanosomes, (ii) tTFIIB accomplishes start site selection in a manner distinct from other TFIIBs, or (iii) start site determination is fulfilled by another factor.
Relevant to this point, it is unclear how start site selection is achieved at human snRNA genes. We note that trypanosome SL RNAs initiate at a conserved 5′ AACU sequence, which is subsequently hypermethylated to form a cap 4 (2
). The complexity of cap formation and start site selection at trypanosome SL RNA gene promoters may require trypanosome-specific proteins in addition to tTBP and tTFIIB.
In summary, our studies of tTFIIB, coupled with previous studies of the variant tSNAPc, tTBP, and RNA polymerase II, contribute to a picture of flexibility inherent to RNA polymerase II-dependent transcription in eukaryotes. Whereas trypanosomes certainly do not carry out transcription in a textbook manner, their mechanisms of transcription may stray only minimally from the established paradigm of RNA polymerase II transcription at snRNA genes. The identification of trypanosomal basal transcription factors and the determination of shared and distinct features from the homologous factors in higher eukaryotes allow us to elucidate the most basic and important requirements for functional RNA polymerase II-dependent transcription.