SummaryNC2 (Dr1/DRAP1) and Mot1p are global repressors of transcription that have been isolated in both Saccharomyces cerevisiae and humans. NC2 is a dimeric histone-fold complex that represses RNA polymerase II transcription through binding to TBP and inhibition of TFIIA and TFIIB. Mot1p is an ATPase that removes DNA-bound TBP upon ATP hydrolysis. In this work, we studied the core promoter specificity of NC2 in vivo using a strain that carries mutated NC2b activity. We show that NC2, like Mot1p, is required for transcription of the HIS3 and HIS4 TATA-less core promoters. Furthermore, whereas neither Mot1p nor NC2 appear to function as repressors of the HIS3 gene in cells growing exponentially in glucose, we find that both are required for repression of the HIS3 TATA promoter when cells go through the diauxic shift. Thus, the activity of these factors is similarly regulated depending upon the physiological conditions, and it appears that core promoters activated or repressed by them in vivo might be distinguishable by whether or not they contain a canonical TATA sequence. Finally, although NC2 is an essential factor for yeast viability, we isolated a mutation in a nonessential component of the holoenzyme, Sin4p, that bypasses the requirement for NC2.
Negative cofactor 2 (NC2) is a dimeric histone‐fold complex that represses RNA polymerase II transcription through binding to TATA‐box‐binding protein (TBP) and inhibition of the general transcription factors TFIIA and TFIIB. Here we study molecular mechanisms of repression by human NC2 in vivo in yeast. Yeast NC2 genes are essential and can be exchanged with human NC2. The physiologically relevant regions of NC2 have been determined and shown to match the histone‐fold dimerization motif. A suppressor screen based upon limiting concentrations of NC2β yielded a cold‐sensitive mutant in the yeast TFIIA subunit Toa1. The single point mutation in Toa1 alleviates the requirement for both subunits of NC2. Biochemical characterization indicated that mutant (mt)‐Toa1 dimerizes well with Toa2; it supports specific recognition of the TATA box by TBP but forms less stable TBP–TFIIA–DNA complexes. Wild‐type but not the mt‐Toa1 can relieve NC2 effects in purified transcription systems. These data provide evidence for a dimeric NC2 complex that is in an equilibrium with TFIIA after the initial binding of TBP to promoter TATA boxes.
A 9-week feeding trial was conducted to investigate the dietary methionine requirement of juvenile Megalobrama amblycephala at a constant dietary cystine level. Six semipurified diets were formulated to contain graded dietary methionine levels from 3.9 to 15.4 g kg À1 in about 2.5 g kg À1 increments in the presence of 2.2 g kg À1 cystine. Results showed that specific growth rate (SGR) and protein efficiency ratio (PER) significantly increased with increasing dietary methionine levels from 3.9 to 12.4 g kg À1 and thereafter kept stable. Maximum protein productive value (PPV), nitrogen retention efficiency (NRE) and liver weight were observed in 8.5 g methionine kg À1 diet. Protein contents in whole fish body were positively correlated with dietary methionine level, while lipid contents were negatively correlated with it. Morphological index and hepatic glutamate-pyruvate transaminase (GPT) activities were independent of dietary methionine levels. However, dietary methionine supplementation significantly improved haematological parameters, plasma methionine and total essential amino acid contents and hepatic glutamate-oxaloacetate transaminase (GOT) activities. Analysis of dose response using broken-line regression on the basis of SGR and PPV versus dietary methionine level estimated the optimum dietary methionine requirements of juvenile M. amblycephala to be between 8.5 and 8.4 g kg À1 of diet (25.0 and 24.7 g kg À1 of protein) in the presence of 2.2 g kg À1 cystine, respectively. Hence, the corresponding total sulphur amino acids requirements of this species were calculated to be 10.7 and 10.6 g kg À1 of diet (31.5 and 31.2 g kg À1 of dietary protein).
A 9-week feeding trial was conducted to estimate the dietary isoleucine requirement of juvenile blunt snout bream. Six isonitrogenous and isoenergetic experimental diets were formulated to contain graded isoleucine levels ranging from 5.3 to 20.1 g kg À1 dry diet. At the end of the experiment, weight gain (WG), specific growth rate (SGR), feed efficiency ratio (FER) and protein efficiency ratio (PER) increased with increasing dietary isoleucine level up to 11.1 g kg À1 dry diet, and dietary isoleucine level above 14.2 g kg À1 dry diet declined these performances. Dietary isoleucine levels (14.2 and 17.3 g kg À1 dry diet) significantly improved whole-body protein content, but decreased whole-body lipid, plasma triglyceride and cholesterol contents. Significantly lower visceral fat index (VFI) in fish fed with 14.2 g kg À1 dietary isoleucine was observed compared to those fed with deficient or excessive isoleucine. Dietary isoleucine supplementation significantly increased plasma isoleucine concentration, while plasma valine and leucine concentrations showed a reversed trend. Dietary isoleucine levels regulated the target of rapamycin (TOR) gene expression and improved plasma superoxide dismutase (SOD) activity in juvenile blunt snout bream. Based on second-order polynomial regression model analysis of SGR and FER, the optimum dietary isoleucine requirement was estimated to be 13.8 g kg À1 dry diet (40.6 g kg À1 dietary protein) and 14.0 g kg À1 dry diet (41.2 g kg À1 dietary protein), respectively.
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