TATA box-binding protein (TBP)-related factor 2 (TRF2), a third member of the TBP family

Abstract

The TATA box-binding protein (TBP) is an essential component of the RNA polymerase II transcription apparatus in eukaryotic cells. Until recently, it was thought that the general transcriptional machinery was largely invariant and relied on a single TBP, whereas a large and diverse collection of activators and repressors were primarily responsible for imparting specificity to transcription initiation. However, it now appears that the "basal" transcriptional machinery also contributes to specificity via tissue-specific versions of TBP-associated factors as well as a tissue-specific TBP-related factor (TRF1) responsible for gene selectivity in Drosophila. Here we report the cloning of a TBP-related factor (TRF2) that is found in humans, Drosophila, Caenorhabditis elegans, and other metazoans. Like TRF1 and TBP, TRF2 binds transcription factor IIA (TFIIA) and TFIIB and appears to be part of a larger protein complex. TRF2's primary amino acid structure suggests divergence in the putative DNA binding domain, and not surprisingly, it fails to bind to DNA containing canonical TATA boxes. Most importantly, TRF2 is associated with loci on Drosophila chromosomes distinct from either TBP or TRF1, so it may have different promoter specificity and regulate a select subset of genes. These findings suggest that metazoans have evolved multiple TBPs to accommodate the vast increase in genes and expression patterns during development and cellular differentiation.

Figure 1

Comparison of TBP family members. (a) Percent identity in the bipartite repeat domain between the Drosophila TBP family members. (b) Organization of domains in TBP family members. The arrows represent the bipartite repeats of the core domain. (c) Alignment of the core domains of dTRF2, hTRF2, ceTRF2, and the TBP consensus sequence, with “X” denoting nonconserved residues. ∗ denotes residues conserved between TRF2 family members that are different from the TBP consensus. The TBP consensus was obtained by comparing Drosophila, human, and C. elegans TBP. Shown are residues 198–379 of dTRF2, 7–186 of hTRF2, and 178–466 of C. elegans TRF2. Full-length Drosophila TRF2 and full-lentgh human TRF2 have been deposited in the GenBank database (accession nos. AF136569 and AF136570). # in the ceTRF2 sequence denotes the 109-residue insertion.

Figure 2

Analysis of dTBP, dTRF1, and dTRF2 conserved residues. (a) Alignment of dTBP, dTRF1, and dTRF2. Red boxes highlight identical residues; a, b, and d indicate TFIIA-, TFIIB-, and DNA-binding residues, respectively (18, 23, 25, 26). (b and c) Location of residues conserved in dTRF1 (a) and dTRF2 (b) visualized on the TBP model. Red residues are conserved with TBP, and blue residues are nonconserved. The TBP model is as described in ref. , with the N-terminal repeat on the right.

Figure 3

Biochemical analysis of TRF2. (a–d) Western blot analysis of TFIIA and TFIIB binding to TRF2. A GST fusion of dTRF2 (a and b) or hTRF2 (c and d) and GST (a–d) was bound to glutathione beads and then incubated with dTFIIA (a), dTFIIB (b), hTFIIA (c), or hTFIIB (d). Unbound protein was then washed away, and the bound protein was detected by Western blot analysis. (e) Elution of endogenous dTRF2 from a Superose 6 gel filtration column (Amersham Pharmacia). Endogenous Drosophila TRF2 eluted slightly after the 670-kDa molecular mass standard. The elution profile of the molecular mass standards is indicated along the axis.

Figure 4

Northern blot analysis of hTRF2 expression in multiple human tissues. (Upper) Blots were probed with TRF2 and actin. An alternative TRF2 transcript was observed in testis (∗), and an alternative actin transcript was observed in both muscle and heart (∗). (Lower) Comparison of relative hTRF2 levels. The hTRF2 signal was normalized against actin, and the testis level was arbitrarily set to 100. pbl, peripheral blood leukocyte.

Figure 5

TRF2 and TAF_II250 localization on Drosophila polytene chromosomes. Anti-TAF_II250 (raised in a mouse) antibodies visualized with Texas red-conjugated secondary antibody is shown in red, and anti-dTRF2 (raised in a rabbit) antibodies visualized with FITC-conjugated secondary antibodies is shown in green. (a) Anti-TAF_II250 only. (b) Both anti-TAF_II250 and anti-dTRF2. (c) Anti-dTRF2 only.