Literature summary for 5.4.99.B22 extracted from

Huet, T.; Miannay, F.A.; Patton, J.R.; Thore, S.
Steroid receptor RNA activator (SRA) modification by the human pseudouridine synthase 1 (hPus1p) RNA binding, activity, and atomic model (2014), PLoS ONE, 9, e94610 .

Search for more literature for 5.4.99.B22

Cloned(Commentary)

Cloned (Comment)	Organism
expression in Escherichia coli	Homo sapiens
gene PUS1, recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3)	Homo sapiens

Crystallization (Commentary)

Crystallization (Comment)	Organism
purified recombinant detagged truncated enzyme mutant . DELTAhPus1p and mutant D146A DELTAhPus1p, sitting drop technique, mixing of 7 mg/ml protein in 20 mM HEPES, pH 7.0, 150 mM NaCl, 5 mM MgCl2, and 5 mM TCEP, with an equal amount of reservoir solution containing for DELTAhPus1p crystals 28% PEG 3350/glycerol, 0.1 M Bicine/Tris, pH 8.5, 10% amino acids mixture (with 0.02 M sodium L-glutamate, 0.02 M DL-alanine, 0.02 M glycine, 0.02 M DL-lysine HCl, 0.02 M DL-serine), and for D146A DELTAhPus1p crystals 17% PEG 8000, and 0.1 M HEPES, pH 8.0, both at 4°C, for 2-3 days, X-ray diffraction structure determination and analysis at 2.0-2.7 A resolution, respectively, molecular replacement using Pus1p structure, PDB ID 4ITS, as a model	Homo sapiens
sitting drop technique. The crystal structure of the catalytic domain of hPus1p and the D146A mutant of the enzyme is determined at 2.0 A resolution, alone and in a complex with several molecules present during crystallisation	Homo sapiens

Crystallization (Comment)

Organism

purified recombinant detagged truncated enzyme mutant . DELTAhPus1p and mutant D146A DELTAhPus1p, sitting drop technique, mixing of 7 mg/ml protein in 20 mM HEPES, pH 7.0, 150 mM NaCl, 5 mM MgCl2, and 5 mM TCEP, with an equal amount of reservoir solution containing for DELTAhPus1p crystals 28% PEG 3350/glycerol, 0.1 M Bicine/Tris, pH 8.5, 10% amino acids mixture (with 0.02 M sodium L-glutamate, 0.02 M DL-alanine, 0.02 M glycine, 0.02 M DL-lysine HCl, 0.02 M DL-serine), and for D146A DELTAhPus1p crystals 17% PEG 8000, and 0.1 M HEPES, pH 8.0, both at 4°C, for 2-3 days, X-ray diffraction structure determination and analysis at 2.0-2.7 A resolution, respectively, molecular replacement using Pus1p structure, PDB ID 4ITS, as a model

Homo sapiens

sitting drop technique. The crystal structure of the catalytic domain of hPus1p and the D146A mutant of the enzyme is determined at 2.0 A resolution, alone and in a complex with several molecules present during crystallisation

Homo sapiens

Protein Variants

Protein Variants	Comment	Organism
D146A	site-directed mutagenesis of truncated enzyme mutant DELTAhPus1p, differences between structures of DELTAhPus1p and DELTAhPus1pD146A are limited to external, poorly conserved loops, which confirm the flexibility of these regions	Homo sapiens
additional information	generation of a truncated hPus1p version encompassing residues 83-394, i.e. DELTAhPus1p. The deleted N- and C-termini are not essential for either the RNA binding or the activity of the hPus1p enzyme	Homo sapiens

Organism

Organism	UniProt	Comment	Textmining
Homo sapiens	Q9Y606	-	-

Purification (Commentary)

Purification (Comment)	Organism
-	Homo sapiens
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity chromatography, tag cleavage by TEV protease, followed by gel filtration, to hoomogeneity	Homo sapiens

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
additional information	pseudouridine synthase assays of hPus1p enzymes tRNA and HP7 SRA RNA substrates, i.e. RNA of the minimal RNA fragment within steroid receptor RNA activator (SRA), termed HP7. Mouse mitochondrial tRNAAsp is used as a positive control for hPus1p activity	Homo sapiens	?	-	?

Subunits

Subunits	Comment	Organism
More	the hPus1p enzyme binds to its RNA substrate as a monomer. The C-terminal end of hPus1p folds into two alpha-helices and these helices pack against the back of the hPus1p molecule, which prevents the dimerization observed in the bacterial homologue, TruA. This C-terminal region is responsible for the atypical RNA substrate binding and activity of hPus1p. Enzyme crystal structure analysis, overview	Homo sapiens

Synonyms

Synonyms	Comment	Organism
hPus1p	-	Homo sapiens

General Information

General Information	Comment	Organism
metabolism	the enzyme modulate class I and class II nuclear receptor responses through its ability to modify the steroid receptor RNA activator	Homo sapiens
additional information	hPus1p active site architecture analysis, overview	Homo sapiens