scholarly journals Gal4 activation domain 9aaTAD could be inactivated by adjacent mini-inhibitory domain and reactivated by distal re-activation domain

2017 ◽  
Author(s):  
Martin Piskacek ◽  
Marek Havelka ◽  
Martina Rezacova ◽  
Andrea Knight
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Small Region ◽  
Inhibitory Effect ◽  
Prediction Algorithm ◽  
Transactivation Domain ◽  
Activation Domain ◽  
Activation Domains ◽  
Gal4 Activation Domain ◽  
Inhibitory Domain

AbstractThe characterisation of the activation domains started three decades ago with Gcn4 and Gal4 activators. The amorphous character of the activation domains strongly hindered their definition. Moreover, during the attempts to localise the Gal4 activation domain, the artificial peptides, an unintended consequence of cloning, were responsible for artificial transcriptional activity of the several Gal4 constructs. These artefacts produced enormous experimental bias and misconception. The presence of inhibitory domains in some Gal4 constructs made the misperception even worse. Previously, we reported that the nine amino acid transactivation domain, 9aaTAD, is the exclusive activation domain in the Gal4 protein. The activation domain 9aaTAD could be identified in Gal4 paralogs Oaf1, Pip2, Pdr1, Pdr3 and other activators p53, E2A and MLL. Surprisingly, the activation domain 9aaTAD was reported as misconception for Gal4 activator. Here we demonstrated that small region of 10 amino acids adjacent to the Gal4 activation domain 9aaTAD is an inhibitory domain, which the authors included in their constructs. Moreover, we identified Gal4 region, which was able to the reverse the inhibitory effect. The 9aaTAD re-activation domain was localized to the 13 amino acid long region. In this report we clarified the numerous confusions and rebutted supposed 9aaTAD misconception.SummaryThe activation domain 9aaTAD has decisive function in Gal4 activation. Gal4 activation domain 9aaTAD could be inhibited by adjacent region of 10 amino acids. The inhibited Gal4 activation domain 9aaTAD could be reactivated by 13 amino acid long Gal4 region. The activation domains 9aaTAD could be identified by our 9aaTAD prediction algorithm, especially in the Gal4 family.

scholarly journals The regulatory domain of human heat shock factor 1 is sufficient to sense heat stress.

1996 ◽  
Vol 16 (3) ◽  
pp. 839-846 ◽  
Author(s):  
E M Newton ◽  
U Knauf ◽  
M Green ◽  
R E Kingston
Keyword(s):  
Amino Acids ◽  
Heat Shock ◽  
Transcriptional Activation ◽  
Heat Shock Factor ◽  
Acid Activation ◽  
Regulatory Domain ◽  
Activation Domain ◽  
Control Temperature ◽  
Activation Domains ◽  
Transcriptional Activation Domains

Heat shock factor (HSF) activates transcription in response to cellular stress. Human HSF1 has a central regulatory domain which can repress the activity of its activation domains at the control temperature and render them heat shock inducible. To determine whether the regulatory domain works in tandem with specific features of the HSF1 transcriptional activation domains, we first used deletion and point mutagenesis to define these activation domains. One of the activation domains can be reduced to just 20 amino acids. A GAL4 fusion protein containing the HSF 1 regulatory domain and this 20-amino-acid activation domain is repressed at the control temperature but potently activates transcription in response to heat shock. No specific amino acids in this activation domain are required for response to the regulatory domain; in particular, none of the potentially phosphorylated serine and threonine residues are required for heat induction, implying that heat-induced phosphorylation of the transcriptional activation domains is not required for induction. The regulatory domain is able to confer heat responsiveness to an otherwise completely heterologous chimeric activator that contains a portion of the VP16 activation domain, suggesting that the regulatory domain can sense heat in the absence of other portions of HSF1.

Amino Acid Transport in a Water-mould: The Possible Regulatory Roles of Calcium and N6-(Δ2-isopentenyl)adenine

1975 ◽  
Vol 53 (9) ◽  
pp. 975-988 ◽  
Author(s):  
Danny P. Singh ◽  
Hérb. B. LéJohn
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Transport ◽  
Osmotic Shock ◽  
Inhibitory Effect ◽  
Transport Systems ◽  
Acid Transport ◽  
Isopentenyl Adenine ◽  
Energy Dependent ◽  
Water Mould

Transport of amino acids in the water-mould Achlya is an energy-dependent process. Based on competition kinetics and studies involving the influence of pH and temperature on the initial transport rates, it was concluded that the 20 amino acids (L-isomers) commonly found in proteins were transported by more than one, possibly nine, uptake systems. This is similar to the pattern elucidated for some bacteria but unlike those uncovered for all fungi studied to date. The nine different transport systems elucidated are: (i) methionine, (ii) cysteine, (iii) proline, (iv) serine–threonine, (v) aspartic and glutamic acids, (vi) glutamine and asparagine, (vii) glycine and alanine, (viii) histidine, lysine, and arginine, and (ix) phenylalanine–tyrosine–tryptophan and leucine–isoleucine–valine as two overlapping groups. Transport of all of these amino acids was inhibited by azide, cyanide, and its derivatives and 2,4-dinitrophenol. These agents normally interfere with metabolism at the level of the electron transport chain and oxidative phosphorylation. Osmotic shock treatment of the cells released, into the shock fluid, a glycopeptide that binds calcium as well as tryptophan but no other amino acid. The shocked cells are incapable of concentrating amino acids, but remain viable and reacquire this capacity when the glycopeptide is resynthesized.Calcium played more than a secondary role in the transport of the amino acids. When bound to the membrane-localized glycopeptide, it permits concentrative transport to take place. However, excess calcium can inhibit transport which can be overcome by chelating with citrate. Calculations show that the concentration of free citrate is most important. At low citrate concentrations (less than 1 mM) in the absence of exogenously supplied calcium, enhancement of amino acid transport occurs. At high concentrations (greater than 5 mM), citrate inhibits but this effect can be reversed by titrating with calcium. Evidently, the glycopeptide acts as a calcium sink to regulate the concentration of calcium made available to the cell for its membrane activities.N6-(Δ2-isopentenyl) adenine (a plant growth 'hormone') and analogues mimic the inhibitory effect of citrate and bind to the glycopeptide as well. Replot data for citrate and N6-(Δ2-isopentyl) adenine inhibition indicate that both agents have no more than one binding constant. These results implicate calcium, glycopeptide, and energy-dependent transport of solutes in some, as yet undefinable, way.

scholarly journals Extensive mutagenesis of a transcriptional activation domain identifies single hydrophobic and acidic amino acids important for activation in vivo.

1997 ◽  
Vol 17 (1) ◽  
pp. 115-122 ◽  
Author(s):  
M B Sainz ◽  
S A Goff ◽  
V L Chandler
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Transcriptional Activation ◽  
Carboxyl Terminus ◽  
Wild Type ◽  
Activation Domain ◽  
Hydrophobic Residues ◽  
Transcriptional Activation Domain ◽  
Acidic Amino Acids

C1 is a transcriptional activator of genes encoding biosynthetic enzymes of the maize anthocyanin pigment pathway. C1 has an amino terminus homologous to Myb DNA-binding domains and an acidic carboxyl terminus that is a transcriptional activation domain in maize and yeast cells. To identify amino acids critical for transcriptional activation, an extensive random mutagenesis of the C1 carboxyl terminus was done. The C1 activation domain is remarkably tolerant of amino acid substitutions, as changes at 34 residues had little or no effect on transcriptional activity. These changes include introduction of helix-incompatible amino acids throughout the C1 activation domain and alteration of most single acidic amino acids, suggesting that a previously postulated amphipathic alpha-helix is not required for activation. Substitutions at two positions revealed amino acids important for transcriptional activation. Replacement of leucine 253 with a proline or glutamine resulted in approximately 10% of wild-type transcriptional activation. Leucine 253 is in a region of C1 in which several hydrophobic residues align with residues important for transcriptional activation by the herpes simplex virus VP16 protein. However, changes at all other hydrophobic residues in C1 indicate that none are critical for C1 transcriptional activation. The other important amino acid in C1 is aspartate 262, as a change to valine resulted in only 24% of wild-type transcriptional activation. Comparison of our C1 results with those from VP16 reveal substantial differences in which amino acids are required for transcriptional activation in vivo by these two acidic activation domains.

Osmoltye and tryptophan receptors controlling gastric emptying in the dog

1976 ◽  
Vol 231 (3) ◽  
pp. 848-853 ◽  
Author(s):  
Stephens ◽  
RF Woolson ◽  
AR Cooke
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Gastric Emptying ◽  
Brush Border ◽  
Inhibitory Effect ◽  
Intestinal Cell ◽  
Acid Transport ◽  
Transport Pathway ◽  
Molar Basis ◽  
Intestinal Receptors

The effect of three monosaccharides, three disaccharides, two dipeptides, combinations of tryptophan with two hexoses, one hexitol, and two amino acids ongastric emptying was studied in dogs to further define the samll intestinal receptors responsive to osmolytes and tryptophan. On a molar basis the disacchardies and dipeptides were almost twice as potent as their respective constituent monosaccharides or amino acids implying that the osmoreceptor is deep to the brush border disaccharidases and cytosol dipeptidases. Tryptophan probably acts by a mechanism different from the osmoreceptor since slowing of gastric emptying by tryptophan was inhibited by methionine which has no effect on a stimulant of the osmoreceptor mechanism. Lysine unlike methionine does not share the neutral amino acid transport pathway with tryptophan. Lysine did not change the inhibitory effect of tryptophan on gastric emptying. This imples that transport of tryptophan into the intestinal cell is necessary for its slowing effect. Glucose and galactose also inhibited the tryptophan effect whereas a nonabsorbed hexitor, mannitol, was without effect. Interference by the hexoses was also probably by competition with tryptophan for transport into the cell. These studies further indicate that the tryptophan receptor is different from the osmoreceptor.

Activation domains of gene-specific transcription factors: are histones among their targets?

2004 ◽  
Vol 82 (4) ◽  
pp. 453-459 ◽  
Author(s):  
Alexandre M Erkine
Keyword(s):  
Transcription Factors ◽  
Amino Acid ◽  
Chromatin Remodeling ◽  
Transcription Initiation ◽  
Protein Complexes ◽  
Amino Acid Sequences ◽  
Gene Promoters ◽  
Activation Domain ◽  
Activation Domains ◽  
Multiple Protein

Activation domains of promoter-specific transcription factors are critical entities involved in recruitment of multiple protein complexes to gene promoters. The activation domains often retain functionality when transferred between very diverse eukaryotic phyla, yet the amino acid sequences of activation domains do not bear any specific consensus or secondary structure. Activation domains function in the context of chromatin structure and are critical for chromatin remodeling, which is associated with transcription initiation. The mechanisms of direct and indirect recruitment of chromatin-remodeling and histone-modifying complexes, including mechanisms involving direct interactions between activation domains and histones, are discussed.Key words: activation domain, transcription, chromatin, nucleosome.

scholarly journals Inhibitory effect of pH5 enzyme from non-lactating bovine mammary gland on various stages of protein synthesis in the rat liver amino acid-incorporating system

1969 ◽  
Vol 115 (4) ◽  
pp. 671-678 ◽  
Author(s):  
M. D. Herrington ◽  
A. O. Hawtrey
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Mammary Gland ◽  
Amino Acid ◽  
Ammonium Sulphate ◽  
Rat Liver ◽  
Inhibitory Effect ◽  
Bovine Mammary Gland ◽  
Free System ◽  
Enzyme Fraction

1. pH5 enzyme from non-lactating bovine mammary gland was found to contain potent inhibitors of protein synthesis in the rat liver cell-free system. These inhibitors affect (a) formation of aminoacyl-tRNA where tRNA represents transfer RNA, (b) transfer of labelled amino acids from rat liver amino[14C]acyl-tRNA to protein in rat liver polyribosomes, and (c) incorporation of 14C-labelled amino acids into peptide by rat liver polyribosomes supplemented with rat liver pH5 enzyme. 2. Increasing amounts of pH5 enzyme from bovine mammary gland progressively inhibited the incorporation of labelled amino acids into protein by a complete incorporating system from rat liver. Approx. 80% inhibition was observed at a concentration of 2mg. of protein of pH5 enzyme from bovine mammary gland. The inhibitory effect of the bovine pH5 enzyme fraction could not be overcome by the addition of increasing amounts of rat liver pH5 enzyme. 3. Fractionation of bovine pH5 enzyme with ammonium sulphate into four fractions showed that all the fractions inhibited the incorporation of 14C-labelled amino acids in the rat liver system, but to varying extents. The highest inhibition observed (90%) was exhibited by the 60%-saturated-ammonium sulphate fraction. 4. Heat treatment of bovine pH5 enzyme at various temperatures caused only a partial loss of its inhibitory effect on labelled amino acid incorporation by the rat liver system. Treatment at 105° for 5min. resulted in the bovine pH5 enzyme fraction losing 30% of its inhibitory activity. 5. pH5 enzyme from bovine mammary gland strongly inhibited the charging of rat liver tRNA in the presence of its own pH5 enzymes. 6. The transfer of labelled amino acids from rat liver amino[14C]acyl-tRNA to protein in a system containing rat liver polyribosomes and pH5 enzyme was almost completely inhibited by bovine pH5 enzyme at a concentration of 2mg. of protein of the enzyme fraction. 7. One of the inhibitors of various stages of protein synthesis in rat liver present in bovine pH5 enzyme was identified as an active ribonuclease, and the second inhibitor present was shown to be tRNA.

TSAO-T Analogues Bearing Amino Acids at Position N-3 of Thymine: Synthesis and Anti-Human Immunodeficiency Virus Activity

2000 ◽  
Vol 11 (1) ◽  
pp. 61-69 ◽  
Author(s):  
C Chamorro ◽  
E De Clercq ◽  
J Balzarini ◽  
M-J Camarasa ◽  
A San-Félix
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Inhibitory Effect ◽  
Acid Moiety ◽  
Hiv Replication ◽  
Virus Activity ◽  
Immunodeficiency Virus ◽  
Amino Acid Moiety ◽  
Anti Hiv ◽  
Hiv 1

Novel analogues of the anti-HIV-1 lead compound [1-[2‘,5’-bis- O-( tert-butyldimethylsilyl)-β-D-ribofuranosyl]thymine]-3‘-spiro-5’-(4“-amino-1”,2“-oxathiole-2‘,2’-dioxide) (TSAO-T) bearing different amino acids at position N-3 of thymine were prepared and evaluated as inhibitors of HIV replication. The synthesis of the target compounds was accomplished by coupling of the appropriate TSAO intermediate with a conveniently protected (L) amino acid in the presence of BOP and triethylamine, followed by depro-tection of the amino acid moiety. Several TSAO derivatives, bearing at N-3 position of the thymine base an L-amino acid retaining the free carboxylic acid, acquired activity against HIV-2, in addition to their inhibitory effect on HIV-1.

scholarly journals The evolution of the 9aaTAD domain in Sp2 proteins: inactivation with valines and intron reservoirs

2019 ◽  
Author(s):  
Martin Piskacek ◽  
Marek Havelka ◽  
Kristina Jendruchova ◽  
Andrea Knight ◽  
Liam P. Keegan
Keyword(s):  
Transcription Factor ◽  
Amino Acid ◽  
Evolutionary History ◽  
Amino Acid Substitutions ◽  
Activation Domain ◽  
Activation Domains ◽  
Transactivation Domains ◽  
Sp1 Transcription Factor ◽  
Chordate Evolution ◽  
Transcription Activators

AbstractThe Sp1 transcription factor has been defined as glutamine-rich activator. The Nine amino acid TransActivation Domains (9aaTAD) have been identified in numerous transcription activators. Here, we identified the conserved 9aaTAD motif in the Sp1 and in all nine members of SP family with broad natural 9aaTAD variations. We showed by the amino acid substitutions that the glutamine residues are completely dispensable for 9aaTADs function. We described the 9aaTAD domains’ origin and evolutionary history. The ancestral Sp2 gene with inactive 9aaTAD has duplicated in early chordates and created new paralogs Sp1, Sp3 and Sp4. We discovered that the accumulation of valines in the 9aaTADs correlated with the domain inactivation. The Sp2 activation domain, whose dormancy have lasted over 100 million years during chordate evolution, enabled later diversification in the Sp1-4 clade, including both repressors and activators. The new paralogs Sp1 and Sp3 activation domains have regained their original activator function by loss of valines in their 9aaTADs.

Control of RNA Synthesis by Amino Acids in Landschutz Ascites Tumor Cells

1974 ◽  
Vol 52 (10) ◽  
pp. 867-876 ◽  
Author(s):  
Paul Jolicoeur ◽  
Fernand Labrie
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Ribosomal Rna ◽  
Rna Synthesis ◽  
Inhibitory Effect ◽  
Amino Acid Starvation ◽  
Cytoplasmic Rna ◽  
Polysomal Mrna ◽  
Deficient Medium ◽  
Synthesis And Processing

Landschutz cells incubated in amino acid-deficient medium for 2.5 h show a markedly reduced incorporation of [3H]uridine into 18 S and 28 S cytoplasmic ribosomal RNA (rRNA) and into 28 S, 32 S, and 36 S nuclear RNA measured during the last 90 min of incubation, whereas the radioactivity associated with 45 S pre-rRNA is not affected. Ten-minute pulse-labeling and 15-min pulse-chase experiments show that amino acid starvation inhibits both the synthesis and processing of 45 S pre-rRNA. Amino acid starvation has no significant effect on the labeling of the nucleotide pools. This effect of amino acids was specific for rRNA since the synthesis of 4 S and 5 S cytoplasmic RNA separated on polyacrylamide gels and of polysomal mRNA analyzed on sucrose gradients was not significantly affected during amino acid starvation. These data also indicate that RNA synthesis is non-coordinated in Landschutz cells. Among the 13 amino acids essential for growth of these cells, arginine and glutamine appear to be mainly responsible for the inhibition of synthesis of 18 S and 28 S rRNA measured during incubation in complete amino acid-deficient medium. The removal of any one of the other amino acids has a small inhibitory effect on the incorporation of [3H]uridine into rRNA and their effect on the synthesis of 18 S rRNA is more pronounced than on that of 28 S rRNA. Such effect results in an unbalanced production of these two ribosomal RNA species.

EFFECTS OF DIHYDROSTREPTOMYCIN ON AMINO ACID INCORPORATION INTO THE PROTEINS OF M. TUBERCULOSIS (BCG)

1959 ◽  
Vol 37 (1) ◽  
pp. 687-697
Author(s):  
E. Stachiewicz ◽  
J. H. Quastel
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Sodium Stearate ◽  
Ehrlich Ascites Carcinoma ◽  
Inhibitory Effect ◽  
Total Radioactivity ◽  
Amino Acid Incorporation ◽  
Acid Incorporation ◽  
Drug Concentrations

A study has been made of the effects of dihydrostreptomycin on amino acid incorporation into the proteins of M. tuberculosis (BCG). Suspensions of this organism on incubation at 37° with glycine-1-C14give rise, aerobically, to labelled proteins in which 80% of the radioactivity appears in the glycine and serine moieties of the proteins and about 20% in alanine and aspartic acid. In presence of glycine-2-C14, radioactivity appears in a larger number of amino acids of the protein. Incubation with serine-3-C14leads to a distribution of radioactivity in the amino acids in BCG proteins but alanine-1-C14and valine-1-C14give rise to proteins with the radioactivity almost entirely in the corresponding amino acids. The process of aerobic incorporation of radioactivity from glycine-1-C14in BCG proteins is stimulated by the presence of glucose, glycerol, sodium pyruvate, sodium stearate, or sodium benzoate in the medium in which the cells are incubated, the rate of incorporation being approximately constant over a period of 4 hours. The incorporation depends largely on the presence of oxygen. Dihydrostreptomycin (33 μg per ml) markedly inhibits labelling of proteins in the cell suspensions in presence of radioactive amino acids, the inhibition increasing with concentration of the streptomycin to an optimal concentration of 200 μg/ml. Penicillin and isonicotinic hydrazide are inactive but chloromycetin is an effective inhibitor. Cyanide, arsenite, and azide are inhibitory. The presence of lecithin stimulates incorporation of radioactivity from glycine-1-C14into BCG proteins. Dihydrostreptomycin inhibitions of amino acid incorporation into BCG proteins increase with time of incubation of the cells with the drug. Concentrations of dihydrostreptomycin that inhibit labelled amino acid incorporation into labelled proteins by 50% have no effect on BCG respiration. The drug has no inhibitory effect on labelled amino acid incorporation in E. coli or Ehrlich ascites carcinoma cells in vitro but is effective with M. phlei. It does not affect selectively the distribution of radioactivities of the component amino acids of BCG proteins; only the total radioactivity incorporated into the proteins is diminished. The results lead to the conclusion that dihydrostreptomycin brings about an inhibition of protein synthesis in the BCG strain of M. tuberculosis at concentrations at which it exerts antibiotic effects.

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