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

Esben Lorentzen,Elena Conti

doi:10.1155/2012/721869

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.

Highlights

RNA exosomes are key players in degradation, processing, and quality control of a wide variety of RNA molecules [1] and have a structurally conserved 9-subunit core common to eukarya and archaea [2]
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
The phosphorolytic activity of the archaeal exosome is reversible resulting in the decay of RNAs in the presence of Pi as well as in the addition of polynucleotide tails in the presence of nucleotide diphosphates, an activity that has been shown to occur in vivo [18, 19]

Summary

Introduction

RNA exosomes are key players in degradation, processing, and quality control of a wide variety of RNA molecules [1] and have a structurally conserved 9-subunit core common to eukarya and archaea [2]. The common exosome core is composed of a hexameric ring of RNase PH subunits (Rrp and Rrp in archaea) capped on one side by three protein subunits containing RNA binding domains (Rrp and Csl in archaea) [3] This architecture results in a barrellike complex with a continuous central channel implicated in RNA binding in both archaea [4] and eukarya [5,6,7]. A number of crystal structures have been determined of archaeal exosomes in both apo and RNA bound forms from Sulfolobus solfataricus [4, 10, 22, 23], Pyrococcus abyssi [24], Archaeoglobus fulgidus [3], and Methanothermobacter thermautotrophicus [25] These structures have revealed the overall architecture of the complexes and visualized RNA at the active site as well as inside the central channel. We present a model for the archaeal exosome highlighting the importance of divalent cations in catalysis

Experimental Procedures

Results and Discussion

RNA I84