PCAF histone acetylase plays a role in regulation of transcription, cell cycle progression, and differentiation. Here, we show that PCAF is found in a complex consisting of more than 20 distinct polypeptides. Strikingly, some polypeptides are identical to TBP-associated factors (TAFs), which are subunits of TFIID. Like TFIID, histone fold-containing factors are present within the PCAF complex. The histone H3- and H2B-like subunits within the PCAF complex are identical to those within TFIID, namely, hTAF(II)31 and hTAF(II)20/15, respectively. The PCAF complex has a novel histone H4-like subunit with similarity to hTAF(II)80 that interacts with the histone H3-like domain of hTAF(II)31. Moreover, the PCAF complex has a novel subunit with WD40 repeats having a similarity to hTAF(II)100.

TFIID is a multiprotein complex composed of TBP and several TAF II s. Small amino-terminal segments (TAF Nterminal domain (TAND)) ofYeast strains containing mutant yTAF II 145 lacking yTANDI or yTANDII showed a temperature-sensitive growth phenotype. The conserved core of dTANDII could substitute for the yTANDII core, and Phe-57 or Tyr-129 described above was critically required for the function of this segment in promoting normal cell growth at 37°C. In these respects, the impact of yTAN-DII mutations on cell growth paralleled their effects on TBP binding in vitro, strongly suggesting that the yTAF II 145-TBP interaction and its negative effects on TFIID binding to core promoters are physiologically important.Transcription of protein coding genes in eukaryotes is carried out by RNA polymerase II and a set of auxiliary initiation factors (1, 2). These factors, including TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH, can be assembled in a combinatorial fashion in vitro to form a preinitiation complex. Recently, it was proposed that most of these factors are preassembled in vivo in the form of holoenzyme and recruited as a single complex to the core promoter to initiate transcription (3, 4). Barberis et al. (5) reported that recruitment of holoenzyme via a fortuitous interaction between GAL4 DNA binding domain and GAL11 or by fusing lexA to GAL11 would suffice for gene activation in Saccharomyces cerevisiae. A similar result was obtained for SRB2, another component of holoenzyme (6). On the other hand, there is evidence that TBP binding to the TATA box is also a rate-limiting step for transcriptional activation that can be accelerated by gene-specific activators (7). In fact, a physical connection of TBP to a DNA binding module bypasses the requirement for activators (8 -10). Given that TBP is a subunit of TFIID and not a component of holoenzyme (11), it appears that recruitment of either TFIID or holoenzyme will suffice for gene activation in yeast (12). However, it is notable that TFIID is required for both cases because mutation of the TATA sequence greatly decreased activation even by holoenzyme recruitment (5).In higher eukaryotes, the question of how activators stimulate transcription has been addressed mostly by biochemical approaches. Particular attention has focused on TFIID, a multiprotein complex composed of TBP and a series of TBP-associated factors (TAF II s), because TAF II s were shown to be indispensable for activated transcription in vitro (13,14). We and others cloned cDNAs encoding TAF II s from various organisms to decipher the molecular basis of transcriptional regulation (15). It is currently known that TAF II s possess some intriguing structural motifs and/or enzymatic activities. For instance, dTAF II 62/dTAF II 42 forms a histone octamer-like heterotetrameric structure (16). dTAF II 230 has multiple enzymatic activities, including a protein serine kinase activity that selectively phosphorylates RAP74 (17) and a histone acetyltransferase activity specific for histones H3 and H4 (18). Furthe...

PCAF histone acetylase is found in a complex with more than 20 associated polypeptides. Here we report cloning and characterization of the 400 kDa PCAF-associated factor referred to as PAF400. PAF400 is almost identical to TRRAP, which binds to c-Myc and E2F, and has significant sequence similarities to the ATM superfamily including FRAP, ATM, ATR, and the catalytic subunit of DNA-PK. Remarkably, PAF400 and FRAP share sequence similarity in broad regions that cover 80% of the entire PAF400 sequence. However, unlike the other members of the ATM superfamily, PAF400 is not a protein kinase as judged from the lack of kinase motif and autophosphorylation activity. We discuss the possibility that PAF400 may play a role in signaling of DNA damage to p53 by stimulation of p53 acetylation.

. 2). An alternative model postulates that the holoenzyme enters the preinitiation complex as a preassembled unit (2, 3). Although the nature of the assembly pathway most relevant to the in vivo context remains unclear, access of TFIID to the core promoter is likely to be a critical step in any pathway, given that only TFIID binds to the promoter in a sequence-specific manner. In support of this view, in vitro recruitment experiments using immobilized promoters demonstrated that the holoenzyme is not recruited independently of TFIID and TFIIA (4).TFIID is a multimeric protein complex consisting of the TATA box-binding protein (TBP) and Ͼ10 distinct TBPassociated factors (TAFs), which are conserved from yeast to man (reviewed in ref. . Consistent with these observations in vivo, TAF-independent activation was demonstrated in transcriptional experiments in vitro, using TFIID-depleted HeLa nuclear extracts, which could possess activator targets other than TAFs (8), or even with a highly purified cell-free transcription system (9, 10). Furthermore, transcription activation is reconstituted by supplementing purified yeast holoenzyme with only TBP (11, 12). These observations suggest that activators contribute to activated transcription by interacting with multiple targets (e.g., basal factors, TAFs, SRB mediators, or other unknown cofactors).Although TAFs are well recognized as positive cofactors, in our view, at least certain TAFs are negative cofactors. Importantly, highly purified TFIID manifests lower transcriptional activity than TBP alone (13) and binds to the core promoter poorly (reviewed in ref. 14). This inhibitory activity for promoter binding in TFIID is suppressed by limited proteolysis of TFIID or by TFIIA (14,15). These results strongly suggest that this inhibitory activity could be intrinsic to TFIID and derived from TAF(s) and is sensitive to proteases. Consistent with this idea, Drosophila (d)TAF230, or the homologous yTAF145, inhibits TBP binding to the TATA box when these TAFs are mixed with TBP in vitro (16)(17)(18). Mutational analyses of dTAF230 indicate that the N-terminal 156 residues inhibit TATA box binding through direct interaction with TBP (19,20). This N-terminal domain (designated as TAND; TAF N-terminal domain), has been dissected into subdomain 1 (dTAND-1; residues 11-77) and subdomain 2 (dTAND-2; residues 82-156), which bind to the concave undersurface and the convex upper surface of TBP in This paper was submitted directly (Track II) to the PNAS office.

Although most patients with spinal muscular atrophy (SMA) are homozygous for deletion of the SMN1 gene, some patients bear one SMN1 copy with a subtle mutation. Detection of such an intragenic mutation may be helpful not only in confirming diagnosis but also in elucidating functional domains of the SMN protein. In this study, we identified a novel mutation in SMN1 of two Japanese patients with type I SMA. DHPLC and sequencing analysis revealed that they harbored a point mutation in SMN1 exon 3, 275G > C, leading to tryptophan-to-serine substitution at amino acid 92 (W92S) at the Nterminal of SMN Tudor domain. In-vitro protein binding assays showed that the mutation severely reduced interaction of the domain with SmB protein and fibrillarin, suggesting that it impairs the critical function of SMN. In conclusion, we reported here that a novel mutation, W92S, in the Tudor domain affects the interaction of SMN with the target proteins.

We isolated a fungal strain HS-1 utilizing either ethylene glycol dibenzoate or ethyl benzoate as sole source of carbon and energy, and identified it as Aspergillus nomius, based on morphological and rDNA analyses. An enzyme hydrolyzing the esters was purified from the culture supernatant of the strain to an electrophoretically homogeneous state. The enzyme was a carboxyl esterase with a monomeric structure, of which the molecular mass was about 60000, and inhibited by phenylmethylsulfonyl fluoride. The enzyme hydrolyzed various benzoate esters and p-nitrophenyl esters, and its hydrolysis rates for the most favorable substrates, n-butyl benzoate and p-nitrophenyl valerate, were comparable.

No abstract

IntroductionIn the past three decades, several methods have been reported for the growth of yeast spheroplasts in liquid culture. When spheroplasts of Saccharomyces cerevisiae were inoculated in rich medium containing 0.4 M KCl as an osmotic stabilizer and cultured for 7 8 h with occasional manual shaking, they entered the fi rst round of mitosis at low cell density. Spheroplasts inoculated at higher cell density showed significant retardation of mitosis Doi, 1979, 1982). Doi and Doi (1982) further showed that spheroplasts underwent the fi rst round of DNA synthesis at roughly the same time as normal cells, but underwent the fi rst round of nuclear division signifi cantly later than normal cells. Spheroplasts cultured in YPG (1% yeast extract, 2% peptone, 2% glucose) medium supplemented with 1 M sorbitol and zymolyase for 18 h increased their diameter threefold, resulting in giant spheroplasts with an average diameter of 13.4 μm (Tamai et al., 1983). Our previous study showed that mitotic nuclear division could be visualized more clearly in giant spheroplasts than in whole cells by DAPI staining and immunofl uorescence microscopy using anti-tubulin antibody Miyakawa, 2000).The formation of giant spheroplasts of microbial cells overcomes the technical problems that are otherwise encountered owing to their small size. Giant spheroplasts of E. coli were successfully generated by spheroplast incubation methods (SI methods) to measure membrane potentials of plasma membrane by a patch-clamp method (Kuroda et al., 1998). Yabe et al. J. Gen. Appl. Microbiol., 57, 177 182 (2011) When spheroplasts of the yeast Saccharomyces cerevisiae are cultured in liquid medium containing osmotic stabilizer, they undergo nuclear division and growth without cell division, resulting in the formation of giant spheroplasts with multinuclei. In this study, we report a simple method for the culture and stable maintenance of giant spheroplasts. The selection of culture media and cell concentration was found to be important for the growth and maintenance of giant spheroplasts. Among the conditions that we tested, static culture in a synthetic Burkholder's medium in 96-well U-bottomed culture plates was most effective. Under appropriate conditions, we could maintain giant spheroplasts for more than 6 days without proliferation of whole cells or marked lysis. The average diameter of spheroplasts can vary from 16 to 53 μm, depending on their initial concentration.