Five genes encoding predicted second homologs of biochemically identified Drosophila embryo TAFs were found encoded in the Drosophila genome by tBlastn searches. In addition to cannonball (can), which encodes a homolog of dTAF5 (Hiller, 2001) the Drosophila genome contains predicted second homologs of several other TAFs: no hitter (nht) (CG15259) a homolog of TAF4, spermatocyte arrest (sa) a homolog of dTAF8 (CG11308), meiosis I arrest (mia) (CG10390) (see also Aoyagi, 2000) a homolog of TAF6 and ryan express (rye) (CG15632) a homolog of TAF12. In addition, the Drosophila genome encodes two previously described TAF10 homologs, dTAF10 and dTAF10b, both expressed during embryogenensis (Hernandez-Hernandez, 2001; Georgieva, 2000; Hiller, 2004 and references therein).
The full-length protein encoded by nht was homologous to the C-terminal portion of dTAF4 and its human homolog hTAF4, but lacked the characteristic N terminal glutamine rich domain. A 924 bp cDNA representing the nht transcript isolated from a testis cDNA library and confirmed by RT-PCR from testis mRNA closely matched the 1 kb size predicted by Northern analysis. Sequencing of this near full length nht cDNA revealed an open reading frame encoding a predicted 245 amino acid protein. Comparison with the genomic DNA sequence revealed no introns in the region covered by the cDNA. The initial methionine was preceded by in frame stop codons in the cDNA, suggesting that it represented the start of the protein. The yeast homolog Mpt1p (yTAF4) also does not contain the extended N-terminal glutamine rich domain (Hiller, 2004).
The C-terminal domain of TAF4 contains a histone fold domain (HFD), a dimerization domain present in several different TAF subunits. Through this characteristic alpha-helix – loop – alpha-helix – loop – alpha-helix structure (alpha1-L1-alpha2-L2-alpha3) several TAFs form specific heterodimer pairs, much like the histones H2A:H2B and H3:H4. Although amino acid sequence conservation was relatively low, the predicted Nht protein contained a domain (aa64-aa114 of nht) that could be aligned with the alpha1-L1-alpha2 region of the histone fold motif of TAF4 and histone H2A. The predicted alignment for the Drosophila TAF4 homologs was checked by threading the amino acid sequences on to the crystal structure obtained for the hTAF4:hTAF12 heterodimer. The dTAF4 sequence fits very well, with an RMSD of 0.14A for the 715-758aa region. The nht sequence also fit into the structure, although the nht sequence was more diverged than dTAF4 from hTAF4 and the RMSD was considerably higher (0.74A, even after optimizing by comparing RMSDs for three different possible local alignments) (Hiller, 2004).
The homology between the predicted nht protein and TAF4 proteins from yeast to humans was particularly striking in the well conserved pattern of alternating hydrophobic and charged or polar residues in the region of alpha helix 2, which forms the core of the interaction domain between TAF4 and its heterodimer partner TAF12. The region of hTAF4 that might contribute to the predicted alpha 3 helix was not included in the crystal structure and is the subject of some debate (Thuault, 2002; Werten, 2002). However, the predicted nht protein did share with the TAF4 homologs from human to yeast a characteristic conserved pattern of hydrophobic, charged and polar residues in the CCTD region near the C terminus of the proteins. In addition, multiple sequence alignments revealed two other extended regions between the HFD and the CCTD that had patterns of charged, polar and hydrophobic residues conserved from nht, to its Drosophila, mosquito, human and C. elegans homologs (Hiller, 2004).
The predicted protein encoded by rye is homologous to TAF12, the histone fold motif-containing binding partner of TAF4. A 550 bp rye cDNA isolated from a testis cDNA library closely matched the 600 bp transcript size estimated from Northern blots. Sequencing of this near full length rye cDNA revealed an open reading frame encoding a 138aa predicted protein with homology to dTAF12. An in frame stop codon located six base pairs upstream of the predicted initial methionine in the cDNA suggested that this corresponded to the start of the protein (Hiller, 2004).
The predicted Rye protein contains a histone fold domain region (amino acid residues 68-130 of rye) that aligns well with the histone fold domain of TAF12 and histone H2B. The sequence of the generally expressed dTAF12 threaded onto the crystal structure of hTAF12 in the hTAF4:hTAF12 dimer well, with an RMSD of 0.09Å for the region from aa91-aa164. Although rye is more diverged, the rye HFD sequence also fit onto the hTAF4:hTAF12 dimer crystal structure (RMSD=0.47). Again, rye and the TAF12 proteins from C. elegans to humans showed a striking pattern of alternating hydrophobic and charged or polar residues, echoing the similar pattern in histone H2B in the region of the predicted HFD, especially in the second helix (Hiller, 2004).
The mia gene encodes a protein homologous to dTAF6. A 2400 bp cDNA isolated from a testis cDNA library corresponded in size to the mia transcript expressed in testis estimated from Northern blots. The testis mia cDNA also contains almost 600 bp of 5' untranslated sequence containing multiple stop codons in all three reading frames upstream of the predicted initial methionine (Hiller, 2004).
The mia N-terminal region resembles corresponding regions of the generally expressed dTAF6 and TAF6 from other organisms, although conservation at the level of amino acid sequence is relatively low. However, multiple sequence alignments reveal that amino acid residues 35-94 of the predicted Mia protein align with the histone fold domain of TAF6 and histone H4 with respect to the pattern of hydrophobic, charged and polar residues. Conservation of this pattern is strongest in the region extending from the middle of the alpha 2 helix through loop 2 and the alpha 3 helix of the HFD, based on the dTAF6:dTAF9 crystal structure (Xie, 1996). In the nucleosome, the regions of alpha 2, loop 2 and alpha 3 of histone H4 are involved both in dimerization, involving residues internal to the H3:H4 pair, and in forming non-symmetrical tetrameric bonds between H4 and H2B via exposed residues (Luger, 1997). The sequence of Mia threaded onto the crystal structure of dTAF6 in the dTAF6:dTAF9 heterodimer with an RMSD of 0.79 for aa33-aa94. In addition to the N-terminal predicted HFD, the Mia protein contains an extended central region (aa176-437 of Mia) with a pattern of hydrophobic, charged, polar and proline residues conserved in TAF6 proteins from yeast to humans. Embedded in this 'TAF6 domain' are several regions of significant amino acid sequence conservation between the predicted Mia protein, dTAF6 and TAF6 from other organisms (Hiller, 2004).
The sa gene encodes a protein homologous to dTAF8, also known as Prodos. Sequencing of an 825 bp sa cDNA isolated by RT-PCR from testis mRNA revealed three protein coding exons separated by two introns (FBgn0037080). The cDNA has an in frame stop codon 84 base pairs upstream of the predicted initial methionine (Hiller, 2004).
The N-terminal region of sa resembles the corresponding regions of the generally expressed dTAF8 (Prodos) and TAF8 from other organisms. Although conservation at the level of amino acid sequence is relatively low, domain searches (PFAM) and multiple sequence alignments suggest that amino acid residues 4-70 of the predicted Sa protein contained a predicted histone fold domain, similar to TAF8 homologs from a variety of species with respect to the pattern of hydrophobic, charged and polar residues. Conservation of this pattern is strongest in the predicted alpha 2 helix region. Although no crystal structure is yet available for TAF8 homologs, secondary structure predictions also suggested an alpha-helix – loop – alpha-helix – loop – alpha-helix pattern in this region of the predicted sa protein, consistent with a histone fold domain structure. In support of the prediction of an N-terminal histone fold domain in dTAF8, deletion of amino acids 1-39 from the generally expressed Drosophila TAF8 homolog Prodos disrupts binding (Hernandez-Hernandez, 2001) between dTAF8 (Prodos) and its binding partner dTAF10B (Hiller, 2004).
The predicted Sa protein also contains an extended central region (aa107-186) with significant amino acid sequence conservation and a pattern of hydrophobic, charged, polar and proline residues conserved in TAF8 homologs from yeast to humans. This region is embedded in a larger domain (aa78-186) listed in the pFam database as pFamB-10670, an unannotated domain characteristic of this family of TAFs (Hiller, 2004).
Because TAF4 and TAF12 physically interact as binding partners through their histone fold domains (Gangloff, 2000; Werten, 2002; Yokomori, 1993), whether the Drosophila testis-specific TAF4 and TAF12 homologs nht and rye also interact structurally was tested using a bacterial co-expression and GST pulldown assay. In bacteria carrying a GST-nht fusion construct encoding full length Nht and an empty vector in place of a Rye-FLAG fusion construct, the GST-nht fusion protein was detected in total bacterial extracts (T) under inducing conditions, but was not soluble in the absence of Rye-FLAG fusion protein. A portion of Rye containing the histone fold region fused to a FLAG epitope was not stable and failed to accumulate when expressed in bacteria in the absence of Nht. However, when both the Nht and Rye fusion proteins were co-expressed in the same bacteria, both the GST-Nht fusion protein and the FLAG-tagged Rye HFD-containing fragment accumulated in the total bacterial extract (T) and were soluble. When the GST-Nht fusion protein was isolated from extracts from bacteria expressing both the Nht and Rye fusion proteins by binding to glutathione-Sepharose, the FLAG-Rye fusion bound and co-eluted with GST-Nht (Hiller, 2004).
The physical interaction between the Nht and Rye fusion proteins appeared to be specific for the testis-specific partners. The C-terminal region of the generally expressed Drosophila TAF4 homolog dTAF4, analogous to the full length Nht protein, interacts with dTAF12: a FLAG epitope-tagged fusion protein containing the histone fold region of dTAF12 is stable and co-purifies with the GST-dTAF4 C-terminal domain fusion protein on glutathione-Sepharose. However, under the same conditions, the C terminal (HFD containing) domain of the generally expressed dTAF4 fused to GST did not stabilize FLAG-tagged Rye when the two were co-expressed in bacteria. In similar assays, the GST-Nht fusion protein was not solubilized when co-expressed with the FLAG-dTAF12 HFD containing fragment, and so was not available to bind to and elute from the glutathione-Sepharose (Hiller, 2004).
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