Cloned (Comment) | Organism |
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The PARN fragment comprising amino acids 445-540 is amplified from a human cDNA library and cloned into the expression vector, the glutathione S-transferase-PARN-RNA-recognition motif fusion protein is expressed in Escherichia coli BL21(DE3). PARN mutants are generated from the wild-type clone (pGEX-6P-1 PARN445-540) using a Site-Directed Mutagenesis Kit. SeMet-containing PARN-RNA-recognition motif is expressed in Escherichia coli. | Homo sapiens |
Crystallization (Comment) | Organism |
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crystal structure of the PARN-RNA-recognition motif domain (residues 445-540) with a bound 7-methylguanosine triphosphate nucleotide shows a remarkable conformational flexibility of the RNA-recognition motif domain, crystal structure is refined at a resolution of 2.1 A, protein folds into a three-stranded antiparallel beta-sheet that is flanked by one alpha-helix connecting beta-strands beta1 and beta2 | Homo sapiens |
Protein Variants | Comment | Organism |
---|---|---|
D478A | generated by site-directed mutagenesis, Kd value for 7-methylguanosine triphosphate is 20.0 microM | Homo sapiens |
K454A | generated by site-directed mutagenesis, Kd value for 7-methylguanosine triphosphate is 20.03 microM | Homo sapiens |
T458A | generated by site-directed mutagenesis, Kd value for 7-methylguanosine triphosphate is 30.58 microM | Homo sapiens |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
additional information | Homo sapiens | PARN interacts not only with the 3'-end of the mRNA but also with its 5'-end as PARN contains an RNA-recognition motif domain that specifically binds both the poly(A) tail and the 7-methylguanosine cap. The interaction of PARN with the 5'-cap of mRNAs stimulates the deadenylation activity and enhances the processivity of this reaction | ? | - |
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Organism | UniProt | Comment | Textmining |
---|---|---|---|
Homo sapiens | O95453 | - |
- |
Purification (Comment) | Organism |
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Cell lysis is performed in a buffer containing 400 mM NaCl, 50 mM Tris/HCl, pH 8.0, 2 mM ethylenediaminetetraacetic acid and 2 mM dithiothreitol. The supernatant of the lysate is loaded onto a glutathione-Sepharose column and is eluted with lysis buffer containing 30 mM reduced glutathione. Glutathione S-transferase-PARN-RNA-recognition motif is incubated with protease and a final gel filtration is performed using a buffer containing 300 mM NaCl, 20 mM Tris/HCl, pH 8.0, and 2 mM DTT. PARN-RNA-recognition motif is concentrated to 8.8 mg/ml using a vivaspin concentrator, and 7-methylguanosine triphosphate is added in 6fold molar excess. SeMet-containing PARN-RNA-recognition motif purification is analogous, with the exception that the DTT concentration is elevated to 5 mM. | Homo sapiens |
Specific Activity Minimum [µmol/min/mg] | Specific Activity Maximum [µmol/min/mg] | Comment | Organism |
---|---|---|---|
additional information | - |
the Kd value of the wild type PARN-RNA-recognition motif enzyme for 7-methylguanosine triphosphate is 6.94 microM | Homo sapiens |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
additional information | PARN interacts not only with the 3'-end of the mRNA but also with its 5'-end as PARN contains an RNA-recognition motif domain that specifically binds both the poly(A) tail and the 7-methylguanosine cap. The interaction of PARN with the 5'-cap of mRNAs stimulates the deadenylation activity and enhances the processivity of this reaction | Homo sapiens | ? | - |
? |
Subunits | Comment | Organism |
---|---|---|
homodimer | gel filtration experiments show that the PARN-RNA-recognition motif domain predominantly exists as a monomer, but about 5% elute at a volume corresponding to a homodimer | Homo sapiens |
monomer | gel filtration experiments show that the PARN-RNA-recognition motif domain predominantly exists as a monomer, but about 5% elute at a volume corresponding to a homodimer | Homo sapiens |
Synonyms | Comment | Organism |
---|---|---|
PARN | - |
Homo sapiens |
poly(A)-specific ribonuclease | - |
Homo sapiens |