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Crystal structure of a 9-subunit archaeal exosome in pre-catalytic states of the phosphorolytic reaction.

Lorentzen E, Conti E - Archaea (2012)

Bottom Line: The RNA exosome is an important protein complex that functions in the 3' processing and degradation of RNA in archaeal and eukaryotic organisms.These structures represent views of precatalytic states of the enzyme and allow the accurate determination of the substrate binding geometries.The high degree of structural conservation between the archaeal exosome and the PNPase including the requirement for divalent metal ions for catalysis is discussed.

View Article: PubMed Central - PubMed

Affiliation: Department of Structural Cell Biology, Max Planck Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany.

ABSTRACT
The RNA exosome is an important protein complex that functions in the 3' processing and degradation of RNA in archaeal and eukaryotic organisms. The archaeal exosome is functionally similar to bacterial polynucleotide phosphorylase (PNPase) and RNase PH enzymes as it uses inorganic phosphate (Pi) to processively cleave RNA substrates releasing nucleoside diphosphates. To shed light on the mechanism of catalysis, we have determined the crystal structures of mutant archaeal exosome in complex with either Pi or with both RNA and Pi at resolutions of 1.8 Å and 2.5 Å, respectively. These structures represent views of precatalytic states of the enzyme and allow the accurate determination of the substrate binding geometries. In the structure with both Pi and RNA bound, the Pi closely approaches the phosphate of the 3'-end nucleotide of the RNA and is in a perfect position to perform a nucleophilic attack. The presence of negative charge resulting from the close contacts between the phosphates appears to be neutralized by conserved positively charged residues in the active site of the archaeal exosome. The high degree of structural conservation between the archaeal exosome and the PNPase including the requirement for divalent metal ions for catalysis is discussed.

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Overall structure of the S. solfataricus exosome with the two RNase PH subunits Rrp41 and Rrp42 displayed in blue and green, respectively, and the RNA binding protein Rrp4 displayed in yellow. The bound RNA substrate is shown as a stick model and the inorganic phosphate atoms as spheres. The picture on the right shows a 90-degree rotation around the horizontal axis with the 3-fold symmetry axis indicated as a triangle.
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fig1: Overall structure of the S. solfataricus exosome with the two RNase PH subunits Rrp41 and Rrp42 displayed in blue and green, respectively, and the RNA binding protein Rrp4 displayed in yellow. The bound RNA substrate is shown as a stick model and the inorganic phosphate atoms as spheres. The picture on the right shows a 90-degree rotation around the horizontal axis with the 3-fold symmetry axis indicated as a triangle.

Mentions: To trap a complex of the archaeal exosome with Pi and RNA substrates bound at the active site, nonameric 3∗(Rrp41/Rrp42/Rrp4) complex from S. solfataricus with the D182A point mutation in the Rrp41 protein (Rrp41D182A) was used. This point mutation was previously shown to completely abolish RNase activity but is not directly involved in RNA or phosphate binding and presumably allows for both substrates to bind without being turned over [10, 22]. Rrp4/Rrp41D182A/Rrp42 mutant exosome crystallized in the cubic space group P213 with one copy of each subunit in the asymmetric unit and the full 9-subunit complex is generated by a crystallographic 3-fold axis (Figure 1). Pi and RNA were soaked into crystals that were cooled in liquid nitrogen at various time points resulting in the determination of a crystal structure of the archaeal exosome at 1.8 Å resolution with Pi bound at the active site and a 2.5 Å resolution structure where both RNA and Pi are bound at the active site (see Table 1 for data collection and refinement statistics).


Crystal structure of a 9-subunit archaeal exosome in pre-catalytic states of the phosphorolytic reaction.

Lorentzen E, Conti E - Archaea (2012)

Overall structure of the S. solfataricus exosome with the two RNase PH subunits Rrp41 and Rrp42 displayed in blue and green, respectively, and the RNA binding protein Rrp4 displayed in yellow. The bound RNA substrate is shown as a stick model and the inorganic phosphate atoms as spheres. The picture on the right shows a 90-degree rotation around the horizontal axis with the 3-fold symmetry axis indicated as a triangle.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3539426&req=5

fig1: Overall structure of the S. solfataricus exosome with the two RNase PH subunits Rrp41 and Rrp42 displayed in blue and green, respectively, and the RNA binding protein Rrp4 displayed in yellow. The bound RNA substrate is shown as a stick model and the inorganic phosphate atoms as spheres. The picture on the right shows a 90-degree rotation around the horizontal axis with the 3-fold symmetry axis indicated as a triangle.
Mentions: To trap a complex of the archaeal exosome with Pi and RNA substrates bound at the active site, nonameric 3∗(Rrp41/Rrp42/Rrp4) complex from S. solfataricus with the D182A point mutation in the Rrp41 protein (Rrp41D182A) was used. This point mutation was previously shown to completely abolish RNase activity but is not directly involved in RNA or phosphate binding and presumably allows for both substrates to bind without being turned over [10, 22]. Rrp4/Rrp41D182A/Rrp42 mutant exosome crystallized in the cubic space group P213 with one copy of each subunit in the asymmetric unit and the full 9-subunit complex is generated by a crystallographic 3-fold axis (Figure 1). Pi and RNA were soaked into crystals that were cooled in liquid nitrogen at various time points resulting in the determination of a crystal structure of the archaeal exosome at 1.8 Å resolution with Pi bound at the active site and a 2.5 Å resolution structure where both RNA and Pi are bound at the active site (see Table 1 for data collection and refinement statistics).

Bottom Line: The RNA exosome is an important protein complex that functions in the 3' processing and degradation of RNA in archaeal and eukaryotic organisms.These structures represent views of precatalytic states of the enzyme and allow the accurate determination of the substrate binding geometries.The high degree of structural conservation between the archaeal exosome and the PNPase including the requirement for divalent metal ions for catalysis is discussed.

View Article: PubMed Central - PubMed

Affiliation: Department of Structural Cell Biology, Max Planck Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany.

ABSTRACT
The RNA exosome is an important protein complex that functions in the 3' processing and degradation of RNA in archaeal and eukaryotic organisms. The archaeal exosome is functionally similar to bacterial polynucleotide phosphorylase (PNPase) and RNase PH enzymes as it uses inorganic phosphate (Pi) to processively cleave RNA substrates releasing nucleoside diphosphates. To shed light on the mechanism of catalysis, we have determined the crystal structures of mutant archaeal exosome in complex with either Pi or with both RNA and Pi at resolutions of 1.8 Å and 2.5 Å, respectively. These structures represent views of precatalytic states of the enzyme and allow the accurate determination of the substrate binding geometries. In the structure with both Pi and RNA bound, the Pi closely approaches the phosphate of the 3'-end nucleotide of the RNA and is in a perfect position to perform a nucleophilic attack. The presence of negative charge resulting from the close contacts between the phosphates appears to be neutralized by conserved positively charged residues in the active site of the archaeal exosome. The high degree of structural conservation between the archaeal exosome and the PNPase including the requirement for divalent metal ions for catalysis is discussed.

Show MeSH
Related in: MedlinePlus