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Cooperative RNP assembly: complementary rescue of structural defects by protein and RNA subunits of archaeal RNase P.

Abstract
Ribonuclease P (RNase P) is a ribonucleoprotein complex that utilizes a Mg(2+)-dependent RNA catalyst to cleave the 5' leader of precursor tRNAs (pre-tRNAs) and generate mature tRNAs. The bacterial RNase P protein (RPP) aids RNase P RNA (RPR) catalysis by promoting substrate binding, Mg(2+) coordination and product release. Archaeal RNase P comprises an RPR and at least four RPPs, which have eukaryal homologs and function as two binary complexes (POP5·RPP30 and RPP21·RPP29). Here, we employed a previously characterized substrate-enzyme conjugate [pre-tRNA(Tyr)-Methanocaldococcus jannaschii (Mja) RPR] to investigate the functional role of a universally conserved uridine in a bulge-helix structure in archaeal RPRs. Deletion of this bulged uridine resulted in an 80-fold decrease in the self-cleavage rate of pre-tRNA(Tyr)-MjaΔU RPR compared to the wild type, and this defect was partially ameliorated upon addition of either RPP pair. The catalytic defect in the archaeal mutant RPR mirrors that reported in a bacterial RPR and highlights a parallel in their active sites. Furthermore, an N-terminal deletion mutant of Pyrococcus furiosus (Pfu) RPP29 that is defective in assembling with its binary partner RPP21, as assessed by isothermal titration calorimetry and NMR spectroscopy, is functional when reconstituted with the cognate Pfu RPR. Collectively, these results indicate that archaeal RPPs are able to compensate for structural defects in their cognate RPR and vice-versa, and provide striking examples of the cooperative subunit interactions critical for driving archaeal RNase P toward its functional conformation.
AuthorsWen-Yi Chen, Yiren Xu, I-Ming Cho, Sri Vidya Oruganti, Mark P Foster, Venkat Gopalan
JournalJournal of molecular biology (J Mol Biol) Vol. 411 Issue 2 Pg. 368-83 (Aug 12 2011) ISSN: 1089-8638 [Electronic] Netherlands
PMID21683084 (Publication Type: Journal Article, Research Support, N.I.H., Extramural, Research Support, U.S. Gov't, Non-P.H.S.)
CopyrightCopyright © 2011 Elsevier Ltd. All rights reserved.
Chemical References
  • Archaeal Proteins
  • Mutant Proteins
  • RNA Precursors
  • RNA, Archaeal
  • Ribonucleoproteins
  • Ribonuclease P
  • Magnesium
Topics
  • Amino Acid Sequence
  • Archaeal Proteins (metabolism)
  • Magnesium (metabolism)
  • Methanococcales (enzymology)
  • Models, Chemical
  • Models, Molecular
  • Molecular Sequence Data
  • Mutant Proteins (metabolism)
  • Mutation
  • Protein Binding
  • Pyrococcus furiosus (enzymology)
  • RNA Precursors (metabolism)
  • RNA, Archaeal (metabolism)
  • Ribonuclease P (metabolism)
  • Ribonucleoproteins (metabolism)
  • Sequence Homology, Amino Acid

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