Structure and Function in Catalytic RNP Assembly

Summary

Principal Investigator: Mark P Foster
Abstract: DESCRIPTION (provided by applicant): The broad, long-term objective of this proposal is to gain structural and dynamic insights into how proteins modulate RNA structure and function in a catalytic ribonucleoprotein (RNP) complex. Ribonuclease P (RNase P) is an essential and ubiquitous ribonucleoprotein enzyme primarily responsible for cleaving the 5'leader sequence during maturation of tRNAs. The eubacterial enzyme is an RNP complex made up of one RNA and one protein subunit. The RNA subunit is catalytic on its own, but the protein is required in vivo. In contrast, several protein subunits with unknown function constitute the human enzyme, and its RNA component alone appears to be inactive. The investigators propose that a principal role of the protein subunits is to stabilize the active conformation of the RNA, and that holoenzyme assembly and substrate recognition proceed via hierarchical mutual binding-induced folding of the protein and RNA subunits. Using the RNase P enzyme from a thermophilic archaebacterium as a model, the investigators will use biochemical and biophysical tools to gain structural insights into the interaction between individual protein components, free and in complex, with the catalytic RNA subunit. The aims are to: 1. Determine the solution structures of one or more protein subunits (or their fragments) by NMR. 2. Map the secondary structure and protein binding sites of the RNA subunit using enzymatic and chemical probes. 3. Characterize protein-protein and protein-RNA interactions in the native and in vitro reconstituted enzyme, and examine whether conformational changes (induced fit) accompany RNP assembly. Successful completion of these aims will enable integration of the data into a three-dimensional model of the RNase P holoenzyme and provide valuable insights into how proteins modulate the enzyme's fUnction. With recent efforts to make use of the substrate selectivity of RNase P to engineer customized ribozymes for use in gene therapy, insights into the molecular basis for the enzyme's function is clearly essential.
Funding Period: 2009-09-30 - 2010-03-31
more information: NIH RePORT

Top Publications

  1. pmc Crystallization and structure determination of the core-binding domain of bacteriophage lambda integrase
    Hari B Kamadurai
    Biophysics Program and Department of Biochemistry, The Ohio State University, Columbus, OH 43201, USA
    Acta Crystallogr Sect F Struct Biol Cryst Commun 64:470-3. 2008
  2. pmc Ligand-induced changes in the structure and dynamics of Escherichia coli peptide deformylase
    Carlos D Amero
    Biophysics Program, The Ohio StateUniversity, Columbus, Ohio 43210, USA
    Biochemistry 48:7595-607. 2009
  3. pmc Discovery of a minimal form of RNase P in Pyrobaculum
    Lien B Lai
    Department of Biochemistry and Center for RNA Biology, Ohio State University, 484 West Twelfth Avenue, Columbus, OH 43210, USA
    Proc Natl Acad Sci U S A 107:22493-8. 2010
  4. pmc Identification of the oriI-binding site of poliovirus 3C protein by nuclear magnetic resonance spectroscopy
    C D Amero
    Department of Biochemistry and Molecular Biology, Pennsylvania State University, 201 Althouse Laboratory, University Park, PA 16802, USA
    J Virol 82:4363-70. 2008
  5. pmc Archaeal/eukaryal RNase P: subunits, functions and RNA diversification
    Nayef Jarrous
    Department of Microbiology and Molecular Genetics, IMRIC, The Hebrew University Hadassah Medical School, Jerusalem 91120, Israel
    Nucleic Acids Res 38:7885-94. 2010
  6. pmc Cleavage of model substrates by archaeal RNase P: role of protein cofactors in cleavage-site selection
    Sylvie Sinapah
    Department of Cell and Molecular Biology, Biomedical Centre, Uppsala University SE 751 24, Uppsala, Sweden
    Nucleic Acids Res 39:1105-16. 2011
  7. pmc An introduction to NMR-based approaches for measuring protein dynamics
    Ian R Kleckner
    The Ohio State University Biophysics Program, 484 West 12th Ave Room 776, Columbus, OH 43210, USA
    Biochim Biophys Acta 1814:942-68. 2011
  8. pmc Tuning riboswitch regulation through conformational selection
    Ross C Wilson
    Department of Biochemistry, Ohio State University, Columbus, OH 43210, USA
    J Mol Biol 405:926-38. 2011
  9. pmc Fidelity of tRNA 5'-maturation: a possible basis for the functional dependence of archaeal and eukaryal RNase P on multiple protein cofactors
    Wen Yi Chen
    Department of Biochemistry, and Center for RNA Biology, The Ohio State University, Columbus, OH 43210, USA
    Nucleic Acids Res 40:4666-80. 2012
  10. pmc Evidence for recycling of external guide sequences during cleavage of bipartite substrates in vitro by reconstituted archaeal RNase P
    I Ming Cho
    Department of Biochemistry, The Ohio State University, Columbus, OH 43210, USA
    J Mol Biol 405:1121-7. 2011

Scientific Experts

  • V Gopalan
  • Mark P Foster
  • L A Kirsebom
  • Nayef Jarrous
  • Lien B Lai
  • Yong Li
  • Wen Yi Chen
  • I Ming Cho
  • Yiren Xu
  • Dileep K Pulukkunat
  • Ross C Wilson
  • Carlos D Amero
  • Ian R Kleckner
  • Hari B Kamadurai
  • Hsin Yue Tsai
  • Sri Vidya Oruganti
  • Sylvie Sinapah
  • Brandon L Crowe
  • William H McClain
  • Christopher J Bohlen
  • Steven A Kawamoto
  • C D Amero
  • Yanglong Zhu
  • Srisunder Subramaniam
  • Hue D Lai
  • Deepali Singh
  • Michael A Stiffler
  • Tina M Henkin
  • Sergei A Kazakov
  • Shiying Wu
  • Angela M Smith
  • Yu Chen
  • B M Fredrik Pettersson
  • Ryan T Fuchs
  • Dwi Susanti
  • Biswarup Mukhopadhyay
  • Amber Simmons
  • Craig A McElroy
  • Douglas W Byerly
  • C E Cameron
  • William P Boomershine
  • Edward J Behrman
  • J J Arnold
  • Christopher G Sudhahar
  • Cynthia L Hatfield
  • Rinku Jain
  • I M Moustafa
  • Jing Sun
  • Walter K Woznick
  • Charles E Bell

Detail Information

Publications26

  1. pmc Crystallization and structure determination of the core-binding domain of bacteriophage lambda integrase
    Hari B Kamadurai
    Biophysics Program and Department of Biochemistry, The Ohio State University, Columbus, OH 43201, USA
    Acta Crystallogr Sect F Struct Biol Cryst Commun 64:470-3. 2008
    ..Thus, this work reveals the domain in the absence of DNA and allows comparison with the DNA-bound form of this catalytically activating domain...
  2. pmc Ligand-induced changes in the structure and dynamics of Escherichia coli peptide deformylase
    Carlos D Amero
    Biophysics Program, The Ohio StateUniversity, Columbus, Ohio 43210, USA
    Biochemistry 48:7595-607. 2009
    ..The results of these studies improve our understanding of the thermodynamic global minimum and have important implications for structure-based drug design...
  3. pmc Discovery of a minimal form of RNase P in Pyrobaculum
    Lien B Lai
    Department of Biochemistry and Center for RNA Biology, Ohio State University, 484 West Twelfth Avenue, Columbus, OH 43210, USA
    Proc Natl Acad Sci U S A 107:22493-8. 2010
    ..This study illustrates an effective combination of next-generation RNA sequencing, computational genomics, and biochemistry to identify a divergent, formerly undetectable variant of an essential noncoding RNA gene...
  4. pmc Identification of the oriI-binding site of poliovirus 3C protein by nuclear magnetic resonance spectroscopy
    C D Amero
    Department of Biochemistry and Molecular Biology, Pennsylvania State University, 201 Althouse Laboratory, University Park, PA 16802, USA
    J Virol 82:4363-70. 2008
    ..Interpretation of our results in the context of an intact oriI provides insight into the architecture of the picornavirus VPg uridylylation complex...
  5. pmc Archaeal/eukaryal RNase P: subunits, functions and RNA diversification
    Nayef Jarrous
    Department of Microbiology and Molecular Genetics, IMRIC, The Hebrew University Hadassah Medical School, Jerusalem 91120, Israel
    Nucleic Acids Res 38:7885-94. 2010
    ....
  6. pmc Cleavage of model substrates by archaeal RNase P: role of protein cofactors in cleavage-site selection
    Sylvie Sinapah
    Department of Cell and Molecular Biology, Biomedical Centre, Uppsala University SE 751 24, Uppsala, Sweden
    Nucleic Acids Res 39:1105-16. 2011
    ..Moreover, our data reveal a hierarchy in recognition elements that dictates cleavage-site selection by archaeal RNase P...
  7. pmc An introduction to NMR-based approaches for measuring protein dynamics
    Ian R Kleckner
    The Ohio State University Biophysics Program, 484 West 12th Ave Room 776, Columbus, OH 43210, USA
    Biochim Biophys Acta 1814:942-68. 2011
    ..This article is part of a Special Issue entitled: Protein Dynamics: Experimental and Computational Approaches...
  8. pmc Tuning riboswitch regulation through conformational selection
    Ross C Wilson
    Department of Biochemistry, Ohio State University, Columbus, OH 43210, USA
    J Mol Biol 405:926-38. 2011
    ..We present a comprehensive model of the structural, thermodynamic, and functional properties of this compact RNA regulatory element...
  9. pmc Fidelity of tRNA 5'-maturation: a possible basis for the functional dependence of archaeal and eukaryal RNase P on multiple protein cofactors
    Wen Yi Chen
    Department of Biochemistry, and Center for RNA Biology, The Ohio State University, Columbus, OH 43210, USA
    Nucleic Acids Res 40:4666-80. 2012
    ..Our results suggest that protein-rich RNase P variants might have evolved to support flexibility in substrate recognition while catalyzing efficient, high-fidelity 5'-processing...
  10. pmc Evidence for recycling of external guide sequences during cleavage of bipartite substrates in vitro by reconstituted archaeal RNase P
    I Ming Cho
    Department of Biochemistry, The Ohio State University, Columbus, OH 43210, USA
    J Mol Biol 405:1121-7. 2011
    ..Taken together, the EGS-based approach merits consideration as a gene knockdown tool in archaea...
  11. pmc Cooperative RNP assembly: complementary rescue of structural defects by protein and RNA subunits of archaeal RNase P
    Wen Yi Chen
    Department of Biochemistry, Ohio State University, Columbus, OH 43210, USA
    J Mol Biol 411:368-83. 2011
    ....
  12. pmc Assembly of the complex between archaeal RNase P proteins RPP30 and Pop5
    Brandon L Crowe
    Department of Biochemistry, Center for RNA Biology, Ohio State University, Columbus, OH 43210 1214, USA
    Archaea 2011:891531. 2011
    ..These results will inform future efforts to elucidate the functional role of the Pop5-RPP30 complex in RNase P assembly and catalysis...
  13. pmc Thermodynamics of coupled folding in the interaction of archaeal RNase P proteins RPP21 and RPP29
    Yiren Xu
    Ohio State Biochemistry Program, The Ohio State University, Columbus, Ohio 43210, United States
    Biochemistry 51:926-35. 2012
    ..These findings provide novel structural and thermodynamic insights into coupled equilibria that allow specificity in macromolecular assemblies...
  14. pmc Dissecting functional cooperation among protein subunits in archaeal RNase P, a catalytic ribonucleoprotein complex
    Wen Yi Chen
    Department of Biochemistry, The Ohio State University, Columbus, OH 43210, USA
    Nucleic Acids Res 38:8316-27. 2010
    ..Collectively, these studies provide new perspectives on the functioning and evolution of an ancient, catalytic ribonucleoprotein...
  15. pmc Ribosomal protein L7Ae is a subunit of archaeal RNase P
    I Ming Cho
    Department of Biochemistry and Molecular Genetics, Center for RNA Biology, Ohio State University, Columbus, OH 43210, USA
    Proc Natl Acad Sci U S A 107:14573-8. 2010
    ....
  16. ncbi Trials, travails and triumphs: an account of RNA catalysis in RNase P
    William H McClain
    Department of Bacteriology, College of Agriculture and Life Sciences, University of Wisconsin, Madison, WI 53706, USA
    J Mol Biol 397:627-46. 2010
    ..Coinciding with the publication of a treatise on RNase P, this review provides a historical narrative, a brief report on our current knowledge, and a discussion of some research prospects on RNase P...
  17. pmc Unexpected diversity of RNase P, an ancient tRNA processing enzyme: challenges and prospects
    Lien B Lai
    Department of Biochemistry and Center for RNA Biology, The Ohio State University, Columbus, OH 43210, USA
    FEBS Lett 584:287-96. 2010
    ....
  18. pmc Functional reconstitution and characterization of Pyrococcus furiosus RNase P
    Hsin Yue Tsai
    Molecular, Cellular and Developmental Biology Graduate Program, Ohio State Biochemistry Program, Ohio State University, Columbus, OH 43210, USA
    Proc Natl Acad Sci U S A 103:16147-52. 2006
    ..Results from our kinetic and footprinting studies on Pfu RNase P, together with insights from recent structures of bacterial RPRs, provide a framework for appreciating the role of multiple Rpps in archaeal RNase P...
  19. pmc Solution NMR of large molecules and assemblies
    Mark P Foster
    Department of Biochemistry, The Ohio State University, 484 West 12th Avenue, Columbus, Ohio 43210, USA
    Biochemistry 46:331-40. 2007
    ..Here we briefly describe the methodological advances that allow NMR spectroscopy of large macromolecules and their complexes and provide a perspective on the wide range of applications of NMR to biochemical problems...
  20. pmc Deciphering RNA structural diversity and systematic phylogeny from microbial metagenomes
    Yanglong Zhu
    Department of Biochemistry and Molecular Biology, and Center for Genetics and Molecular Medicine, School of Medicine, University of Louisville, Louisville, KY 40202, USA
    Nucleic Acids Res 35:2283-94. 2007
    ..e. some archaeal P RNAs have only one nucleotide in the J2/3 joint. We also discuss the phylogenetic analysis based on covariance model of P RNA that offers a few advantages over the one based on 16S rRNA...
  21. pmc Trans cooperativity by a split DNA recombinase: the central and catalytic domains of bacteriophage lambda integrase cooperate in cleaving DNA substrates when the two domains are not covalently linked
    Srisunder Subramaniam
    Department of Biochemistry and Biophysics Program, The Ohio State University, 484 West12th Ave, Columbus, OH 43210, USA
    J Mol Biol 370:303-14. 2007
    ....
  22. pmc Studies on the mechanism of inhibition of bacterial ribonuclease P by aminoglycoside derivatives
    Steven A Kawamoto
    Department of Biochemistry, The Ohio State University, Columbus, OH 43210, USA
    Nucleic Acids Res 36:697-704. 2008
    ....
  23. pmc Studies on Methanocaldococcus jannaschii RNase P reveal insights into the roles of RNA and protein cofactors in RNase P catalysis
    Dileep K Pulukkunat
    Ohio State Biochemistry Program and Department of Biochemistry, The Ohio State University, Columbus, OH 43210, USA
    Nucleic Acids Res 36:4172-80. 2008
    ..Our results suggest a common RNA-mediated catalytic mechanism in all RNase P and help uncover parallels in RNase P catalysis hidden by plurality in its subunit make-up...
  24. pmc Solution structure of Pyrococcus furiosus RPP21, a component of the archaeal RNase P holoenzyme, and interactions with its RPP29 protein partner
    Carlos D Amero
    Biophysics Program, Center for RNA Biology, The Ohio State University, Columbus, Ohio 43210, USA
    Biochemistry 47:11704-10. 2008
    ..The data show that the primary contact with RPP29 is localized to the two helices of RPP21. This information represents a fundamental step toward understanding structure-function relationships of the archaeal RNase P holoenzyme...
  25. pmc Solution structure of an archaeal RNase P binary protein complex: formation of the 30-kDa complex between Pyrococcus furiosus RPP21 and RPP29 is accompanied by coupled protein folding and highlights critical features for protein-protein and protein-RNA in
    Yiren Xu
    Ohio State Biochemistry Program, Center for RNA Biology, The Ohio State University, Columbus, OH 43210, USA
    J Mol Biol 393:1043-55. 2009
    ..These findings provide valuable new insights into mechanisms of RNP assembly and serve as important steps towards a three-dimensional model of this ancient RNP enzyme...
  26. pmc Structure of Pfu Pop5, an archaeal RNase P protein
    Ross C Wilson
    Ohio State Biochemistry Program, Department of Biochemistry, Ohio State University, Columbus, OH 43210, USA
    Proc Natl Acad Sci U S A 103:873-8. 2006
    ..NMR spectra have been used to map the interaction of Pop5 with Pfu Rpp30. The data presented permit tantalizing hypotheses regarding the role of this protein subunit shared by archaeal and eukaryotic RNase P...