Isolation and characterization of a cDNA encoding a novel human transcription factor TFIID subunit containing similarities with histones H2B and H3

Using the yeast two-hybrid system, we isolated a human cDNA that encodes a protein (hp22) interacting with TATA box-binding factor TFIID subunit p80 containing similarity with histone H4. Sequence analysis showed that the open reading frame (ORF) specifies a 161-amino-acid (aa) polypeptide homologous to Drosophila melanogaster TFIID subunit p22 (dp22). Comparison of the aa sequence of human TFIID subunit p22 (hp22) with that of dp22 revealed that p22 is composed of two distinct regions; the less conserved N-terminal (20% identity) and the highly conserved C-terminal (65% identity) regions. Additionally, the C-terminal region was found to contain similarities with histones H2B and H3. Northern blot analysis showed mRNA corresponding to hp22 to be expressed in all tissues examined     

Core promoter binding by histone-like TAF complexes

A major function of TFIID is core promoter recognition. TFIID consists of TATA-binding protein (TBP) and 14 TBP-associated factors (TAFs). Most of them contain a histone fold domain (HFD) that lacks the DNA-contacting residues of histones. Whether and how TAF HFDs contribute to core promoter DNA binding are yet unresolved. Here we examined the DNA binding activity of TAF9, TAF6, TAF4b, and TAF12, which are related to histones H3, H4, H2A, and H2B, respectively. Each of these TAFs has intrinsic DNA binding activity adjacent to or within the HFD. The DNA binding domains were mapped to evolutionarily conserved and essential regions. Remarkably, HFD-mediated interaction enhanced the DNA binding activity of each of the TAF6-TAF9 and TAF4b-TAF12 pairs and of a histone-like octamer complex composed of the four TAFs. Furthermore, HFD-mediated interaction stimulated sequence-specific binding by TAF6 and TAF9. These results suggest that TAF HFDs merge with other conserved domains for efficient and specific core promoter binding.     

The Rad51/RadA N-terminal domain activates nucleoprotein filament ATPase activity

Proteins in the RecA/RadA/Rad51 family form helical filaments on DNA that function in homologous recombination. While these proteins all have the same highly conserved ATP binding core, the RadA/Rad51 proteins have an N-terminal domain that shows no homology with the C-terminal domain found in RecA. Both the Rad51 N-terminal and RecA C-terminal domains have been shown to bind DNA, but no role for these domains has been established. We show that RadA filaments can be trapped in either an inactive or active conformation with respect to the ATPase and that activation involves a large rotation of the subunit aided by the N-terminal domain. The G103E mutation within the yeast Rad51 N-terminal domain inactivates the filament by failing to make proper contacts between the N-terminal domain and the core. These results show that the N-terminal domains play a regulatory role in filament activation and highlight the modular architecture of the recombination proteins.     

Calcium-dependent interaction of annexin I with annexin II and mapping of the interaction sites

Annexins are multifunctional intracellular proteins with Ca²⁺- and phospholipid-binding properties. Their structures consist of four conserved repeat domains that form the core and a diverse N-terminal tail, from which their functional differences may arise. We searched for cellular proteins that interact with the N-terminal tail plus domain I of annexin I (ANX1) by using the yeast two-hybrid method. Screening of a HeLa cell cDNA library yielded annexin II (ANX2) cDNA. The interaction between ANX1 and ANX2 also occurred in vitro in a Ca²⁺-dependent manner. Mapping of the interaction sites revealed that interaction between domain I of ANX1 and domain IV of ANX2 was stronger than the other combinations.     

Dimerization of the type 4 cAMP-specific phosphodiesterases is mediated by the upstream conserved regions (UCRs)

The cAMP-specific PDE4 family consists of four genes, each expressed as several splice variants. These variants are termed long and short forms depending on the presence or absence of two unique N-terminal domains called upstream conserved regions 1 and 2 (UCR1 and 2). UCR1 and UCR2 have been shown to form a module necessary for the activation of PDE4 upon phosphorylation by the cAMP-dependent kinase (PKA). Here we have uncovered PDE4 oligomerization as a novel function for the UCR1/UCR2 module. Using several different approaches including gel filtration, sucrose density gradient centrifugation, pull-down of differentially tagged PDE constructs, and yeast two-hybrid assay, we show that the long PDE4 splice variant PDE4D3 behaves as a dimer, whereas the short splice variant PDE4D2 is a monomer. Internal deletions of either the C-terminal portion of UCR1 or the N-terminal portion of UCR2 abolishes dimerization of PDE4D3 indicating that both domains are involved in this intermolecular interaction. The dimerization, however, is structurally distinguishable from a previously described intramolecular interaction involving the same domains. PKA phosphorylation and site-directed mutagenesis shown to ablate the latter do not interfere with dimerization. Therefore, dimerization of the long PDE4 forms may be an additional function of the UCR domains that further explains differences in the regulatory properties between the long and short PDE4 splice variants.     

Incorporation of the Arc1p tRNA-binding domain to the catalytic core of MetRS can functionally replace the yeast Arc1p-MetRS complex

The catalytic core of methionyl-tRNA synthetase (MetRS) is conserved among all life kingdoms but, depending on species origin, is often linked to non-catalytic domains appended to its N- or C-terminus. These domains usually contribute to protein-protein or protein-tRNA interactions but their exact biological role and evolutionary purpose is poorly understood. Yeast MetRS contains an N-terminal appendix that mediates its interaction with the N-terminal part of Arc1p. Association with Arc1p controls the subcellular distribution of MetRS. Furthermore, the C-terminal part of Arc1p harbors a conserved tRNA-binding domain (TRBD) required for the Arc1p-dependent stimulation of the catalytic activity of MetRS. The same TRBD is found directly fused to catalytic domains of plant and nematode MetRS as well as human tyrosyl-tRNA synthetase. To investigate the purpose of Arc1p-MetRS complex formation in yeast, we tested the ability of TRBD to assist the function of MetRS independently of Arc1p. We attached the TRBD directly to the C-terminus of the MetRS catalytic core (MC) by constructing the chimeric protein MC-TRBD. The effect of MC-TRBD expression on yeast cell growth as well as its localization and in vitro aminoacylation activity were analyzed and compared to that of MC alone or wild-type MetRS, both in the absence or presence of Arc1p. We show that MC-TRBD exhibits improved enzymatic activity and can effectively substitute the MetRS-Arc1p binary complex in vivo. Moreover, MC-TRBD, being exclusively cytoplasmic, also mimics the MetRS-Arc1p complex in terms of subcellular localization. Our results suggest that the sole role of the N-terminal appended domain of yeast MetRS is to mediate the indirect association with the TRBD, which, nevertheless, can also function effectively in vivo when directly fused to the catalytic MetRS core.