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INTRODUCTION

Structure of the nuclear exosome component Rrp6p reveals an interplay between the active site and the HRDC domain. Søren F. Midtgaard*†, Jannie Assenholt†, Anette Thyssen Jonstrup*†, Lan B. Van*†, Torben Heick Jensen†, and Ditlev E. Brodersen*†‡

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INTRODUCTION

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  1. Structure of the nuclear exosome component Rrp6p reveals an interplay between the active site and the HRDC domain Søren F. Midtgaard*†, Jannie Assenholt†, Anette Thyssen Jonstrup*†, Lan B. Van*†, Torben Heick Jensen†, and Ditlev E. Brodersen*†‡ *Centre for Structural Biology, Department of Molecular Biology, University of Aarhus, Gustav Wieds Vej 10c, DK-8000 Aarhus C, Denmark; and †Centre formRNP Biogenesis and Metabolism, Department of Molecular Biology, University of Aarhus, C. F. Møllers Alle´ , bygn. 130, DK-8000 Aarhus C, Denmark PNAS., Vol. 103, pp.11898-11903, 2006 楊哲權

  2. INTRODUCTION Rrp47 3 additional proteins to yeast nuclear core exosome: Rrp6p (human PM/Scl-100):RNase D-type enzyme Mtr4p : DEAD-box RNA helicase Rrp47 : less well characterized Trends Biochem Sci., 2002,27,11-18.

  3. The nuclear exosome is essential for maturation of eukaryotic ribosomal RNAs. Trends Cell Biol., 2002,12,90-96.

  4. Many small nucleolar RNAs (snoRNAs) depend on the nuclear exosome during their maturation. Deletion of Rrp6p in yeast also leads to accumulation of extended forms of both polycistronic snoRNAs and the independently transcribed snoRNAs, The EMBO Journal ,1999,18,5399–5410. Mol. Cell. Biol.,, 2000, 20, 441-452.

  5. RNase D domain Domain structure of yeast Rrp6p RNase D domain : similar to bacterial RNase D, DEDD nucleases, DNA/RNA DEDD nucleases : four conserved acidic residues required for nucleic acid degradation in the 3’-5’ direction (ex. PARN, Klenow fragment ), two divalent metal ion, DEDDy subgroup HRDC domain : helicase and RNase D C-terminal domain, a putative nucleic acids binding domain, specificity N-terminal domain : absent in bacterial RNase D

  6. METHODS AND RESULTS Protein construct used in crystallization : 129-536 (Y361A) Overall structure:

  7. Rrp6p Requires both Mn2+ and Zn2+ to Bind Nucleotides.

  8. Binding of a nucleotide in the active site is associated with a closure of the active-site cleft. Rrp6p Rrp6p-AMP

  9. Recognition of the 3’ Ribonucleotide.

  10. RNA Processing by Rrp6p Is Affected by N-Terminal Anchoring of the HRDC Domain. D457A mutation rendered Rrp6p incapable of proper 3'-end trimming of stable RNAs while retaining the exonucleolytic activity. The N-terminal-HRDC interaction

  11. The importance of the D457–Q133/N142 interaction on Rrp6p RNA-processing efficiency. Correct anchoring of the HRDC domain is required to maintain efficient processing of some substrates by Rrp6p but that the defects in RNA processing are not rate-limiting for growth.

  12. SUMMARY AND DISCUSSION 1. The only characterized structure of a eukaryotic exosome component. 2. Structural homology to the prokaryotic RNase D enzyme. N-terminal extension : may participate in anchoring of Rrp6p to core exosome.

  13. 3. Substrate specificity : H241 ( N-terminal domain ) K342 ( RNase D domain ) 4. The exonuclease-HRDC contact affects processing of some substrates by Rrp6p, such as snR40.

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