Progress 10/01/03 to 09/30/09
Outputs
OUTPUTS: The results of this investigations, and the associated collaborative work in related systems, has resulted in the publication of 11 peer-reviewed papers during the time-frame of this project, and at least two additional upcoming papers. This work has been presented in regional, national and international meetings in both poster and oral formats. In addition, RNA alignments and structures generated during the course of this work are posted on the RNase P Database, our freely-accessible online database. All novel sequences have been submitted to GenBank. PARTICIPANTS: James W. Brown (PI/PD) J. Christopher Ellis (Ph.D. student) John Gantt (M.S. student) Jeff Barnes (M.S. student) Mei-Ping Cheng (postdoc) Jennifer Lowther (B.S. student) Jessica Anderson (B.S. student) Maggie Titus (B.S. student) James Wilde (B.S. student) Jennifer Cockerham (B.S. student) Bobby Freeze (B.S. student) Beatrice Criveanu (B.S. student) Annie Howell (B.S. student) Chris Cardwell (B.S. student) Ayeshia Beavers (B.S. student) Danielle McLauren (B.S. student) Magan Hanna (B.S. student) Alina Lotstein (B.S. student) Jennifer Massingill (B.S. student) Katrina Harvey (B.S. student) Maso Nour (B.S. student) Ashley Cole (B.S. student) Valerie Yager (B.S. student) Xiao Song (B.S. student) Jing Li (M.S. student) Elizabeth Tran (collaborator) E. Suart Maxwell (collaborator) Daniel WIlliams (Ph.D. student) Thomas Hall (Ph.D. student) Steve Marquez (collaborator J. Kirk Harris (collaborator) Norman Pace (collaborator) Ed Breidtschwerdt (collaborator) Christian Pitulle (collaborator) Neocles Leontis (collaborator) Russ Altman (collaborator) Helen Berman (collaborator) David Engelke (collaborator) Steve Harvey (collaborator) Steve Holbrook (collaborator) Fabrice Jossinet (collaborator) Francois Major (collaborator) David Matthews (collaborator) Jane Richardson (collaborator) Jamie Williamson (collaborator) Eric Westhof (collaborator) Doris BEtancourt (collaborator) Telissa Loveless (collaborator) Paul Bishop (collaborator) Amanda Birmingham (collaborator) Paul Griffiths (collaborator) Ryn Kachouri (collaborator) Franz Lang (collaborator) Rob Knight (collaborator) Gerhard Steger (collaborator) Jesse Stombaugh (collaborator) TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: The reason this project has been allowed to terminate is that the direction of the research has become increasingly unrelated to the ARS goals. We have become increasingly computational, and essentially have dispensed with laboratory work and is no longer specifically related to methanogens or methanogenesis - i.e. those organisms important in waste management that were the reason for the ARS project.
Impacts
We have identified the essential protein components of RNase P RNA in Methanothermobacter thermoautotrophicus, and most recently shown that an additional protein (Mth1483p) thought by some to be a subunit of RNase P is neither associated with nor contributes to the catalytic activity of RNase P. Each of these RNase P proteins are homologous to eukaryotic nuclear RNase P proteins. We have determined the protein-protein interactions between these subunits, and these closely mirror those of the yeast nuclear enzyme. Our results on the archaeal RNase P proteins has allowed those working in the eukaryotic nuclear system to focus their attention on he 4 "core" proteins that are sharing between these Kingdoms. They have shown that these proteins are, indeed, the most critical for function, and that subsets of these proteins are all that is required to reconstitute low levels of native catalytic function in vitro.
Publications
- Brown JW, Birmingham A, Griffiths PE, Jossinet F, Kachouri-Lafond R, Knight R, Lang BF, Leontis N, Steger G, Stombaugh J and Westhof E. 2009 The RNA structure alignment ontology. RNA Pubmed ID 19622678
- Ellis JC and Brown JW. 2009 Rfam Section: The RNase P RNA family. RNA Biol. Pubmed ID 19738420
- Ellis JC, Barnes J, and Brown JW. 2007 Is Alba an RNase P subunit RNA Biology 4:169-172 Pubmed ID 18347433
- O'Rourke, L.G., Pitulle, C., Hegarty, B.C., Kraycirik, S., Killary, K.A., Grosenstein, P., Brown, J,W., and Breitschwerdt, E.B. 2005 Bartonella quintana in cynomolgus monkey (Macaca fascicularis). Emerg Infect Dis. 11:1931-1934 PubMed ID 16485482
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Progress 10/01/07 to 09/30/08
Outputs
OUTPUTS: Activities: Three undergraduates were mentored in the course of this research, either on a volunteer basis or for course credit. One graduate rotation student was mentored. Events: This work was presented at the NC ASM meeting. Services: none Products: The RNase P Database, an online web resource. PARTICIPANTS: Students at NCSU: Valerie Yager, Ashley Cole, Gwyn Rowland Naso Nour and Yi-Jian Huang. Collaboratiors on RNA ontology/sequence alignment project: Amanda Birmingham (Thermo Fisher Scientific), Paul E. Griffiths (Department of Philosophy and Centre for the Foundations of Science, University of Sydney), Fabrice Jossinet (Architecture et reactivite de l'ARN, Universite de Strasbourg, Institut de Biologie Moleculaire et Cellulaire du CNRS), Rym Kachouri-Lafond (Architecture et reactivite de l'ARN, Universite de Strasbourg, Institut de Biologie Moleculaire et Cellulaire du CNRS), Rob Knight (Department of Chemistry & Biochemistry, University of Colorado at Boulder), B. Franz Lang (Centre Robert Cedergren, Departement de Biochimie, Universite de Montreal), Neocles Leontis (Department of Chemistry and Center for Biomolecular Sciences, Bowling Green State University), Gerhard Steger (Institut fur Physikalische Biologie, Heinrich-Heine-Universitat Dusseldorf), Jesse Stombaugh (Department of Chemistry and Center for Biomolecular Sciences, Bowling Green State University), Eric Westhof (Architecture et reactivite de l'ARN, Universite de Strasbourg, Institut de Biologie Moleculaire et Cellulaire du CNRS, Strasbourg). Collaborators on Pyrobaculum RNase P project: Todd Lowe (UC-Santa Cruz), Venkat Gopalan (The Ohio State University) and their students. Collaborators on diazotroph work (see publications): Telisa Loveless (NCSU/USDA), Doris Betancourt (NIEHS). TARGET AUDIENCES: The target audiences for this work are the scientists in the fields of RNA structure and function (RNase P in Pyrobaculum work) and molecular bioinformatics as well (RNA alignment work). PROJECT MODIFICATIONS: Our work on RNase P has always had a significant computational component. This component has come to predominate our efforts, and this trend is likely to continue in the future to the point of entirely displacing our "wet lab" work.
Impacts
Change in knowledge: There are two major new insights stemming from our work. The first is a new ontological paradigm for RNA structure alignments, which should be applicable to any macromolecular sequence/structure alignment, in which "correspondence" is assigned only where specified (instead of implicitly and indiscriminately, as columns) and to any defined element of structure rather than just nucleotides (residues). The second is the identification of an extremely reduced RNase P RNA in species of the genus Pyrobaculum, in which the RNA consists essentially of just the catalytic domain, n the specificity domain is absent. This is probably related to unusual tRNA processing in these organisms because pre-tRNA transcripts appear to be transcribed with very short leaders, usually only 1-3 nucleotides. Change in action: Our previous work on archaeal RNase P has focused study on eukaroytic RNase P on the 4 conserved proteins these share. Change in conditions:
Publications
- Betancourt DA, Loveless TM, Brown JW, and Bishop PE. 2008 Characterization of diazotrophs containing Mo-independent nitrogenases isolated from diverse environments. Appl Environ Microbiol 74:3471-3480 (Pubmed ID 18378646)
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Progress 10/01/06 to 09/30/07
Outputs
OUTPUTS: The results of this work have been disseminated (other than in publications) in the form of presentations at scientific meetings: 1) The Need for an RNA Alignment Ontology. A talk given at the RNA Ontology Consortium meeting, May 28-29, 2007, Madison, WI. 2) RNase P in Archaea: Looking to a warm place for P. A talk given at UNC-Chapel Hill Department of Microbiology seminar, September 7, 2007, Chapel Hill, NC 3) RNase P in Pyrobaculum? A poster given at the NC ASM meeting, October 12, 1996, Raleigh, NC 4) RNase P in Pyrobaculum? A poster given at the NC Undergraduate Research Symposium, November 18, 1996, Raleigh, NC 5) Should Azomonas insignis be reclassified? A poster given at the Beijing Youth Science Creation Competition, March 2007, Beijing, China 6) Phylogenetic analysis of Azomonas insignis. A poster given at the NC ASM 2006 Branch Meeting, October 12, 2006, Raleigh, NC
PARTICIPANTS: James W. Brown - PI J. Chris Ellis - Ph.D. student Chris Caldwell - undergraduate student Ayeshia Beavers - undergraduate student Danielle McLauren - undergraduate student Megan Hanna - undergraduate student Alina Lotstein - undergraduate student Jennifer Massingill - undergraduate student Katrina Garvey - undergraduate student Valerie Yager - undergraduate student Informal collaboration with Todd Lowe (UCSC) & Ventkat Gopalan (OSU) on the RNase Ps of Pyrobaculum species. Formal collaboration with Eric Westhof, Fabrice Jossinet & Rym Kachouri (CNRS, Strasborg), Fran Lang (U. Montreal), Neocles Leontis & Jesse Stombagh (BGSU), and Rob Knight (U CO) on RNA ontology.
TARGET AUDIENCES: The scientific community (via talks, seminar & informal connections). This work is incorporated a various levels in our Microbial Diversity course of ca. 50 undergraduate students at NCSU.
PROJECT MODIFICATIONS: Our work has transitioned from a focus on the RNase P of Methanocaldococcus jannaschii to that of Pyrobaculum species because of the more extreme losses of RNA elements in these RNAs. In addition, we are largely broadening the scope of the work to deal with broader issues of RNA comparative informatics as part of the RNA Ontology Consortium.
Impacts
Interest in ribonuclease P generally, and the archaeal enzyme in particular, has three primary aspects. The first is that ribonuclease P is a catalytic RNA, and is apparently a left-over of the early RNA World, and the archaeal system is apparently the most primative of these. Secondly, ribonuclease P is a great potential target for the development of new antimicrobials. Lastly, biophysicists generally, and most specifically in the field of RNA, have flocked to archaeal systems because of their simplicity and the relative ease of biophysical analysis of the RNAs, proteins, and RNA:protein complexes from these organisms.
Publications
- Leontis, N.B., Altman, R., Berman, H., Brenner, S.E., Brown, J.W., Engelke, D., Harvey, S.C., Holbrook, S.R., Jossinet, F., Lewis, S.E., Major, F., Methews, D.H., Richardson, J., Williamson, J.R., and Westhof, E. 2006 The RNA Ontology Consortium: An open invitation to the RNA community. RNA 12:533-541
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Progress 10/01/04 to 09/30/05
Outputs
The first goal of this project is to identify all of the subunits of RNase P in Methanothermobacter thermoautotrophicus, in specific to test whether or not Mth1483p is a subunit of RNase P, and to determine the structures of these proteins (presumably in collaboration). Toward this goal, we have previously determined in preliminary experiments that Mth1483p is not physically associated with RNase P nor is it required (or beneficial) for enzymatic activity in vitro; i.e. it is apparently not an RNase P subunit, and we have been working to substantiate this. Several groups worldwide are determining the structures of these proteins by NMR spectroscopy and X-ray diffraction; most have been solved. The second goal, the determination of the interactions between these proteins, is complete for the subunits we have identified. The third goal, the same as the first two except in Methanocaldococcus jannaschii, we have produced antisera against all of the 4 known presumptive
RNase P proteins (homologs of the 4 necessary and sufficient proteins in M. thermoautotrophiccus) and shown them to be associated with RNase P activity. We previously identified a number of additional potential RNase P protein subunits in M.jannaschii, but have found no evidence that any are associated with enzymatic activity. We are currently working to recapitulate the reconstitution of holoenzyme from recombinant subunits in the M.thermoautotrophicus system and also in M. jannaschii to investigate the role of any additional proteins in the type M enzymes (such as M.jannaschii) and the differences between type A and type M substrate recognition.
Impacts
Interest in ribonuclease P generally, and the archaeal enzyme in particular, has three primary aspects. The first is that ribonuclease P is a catalytic RNA, and is apparently a left-over of the early RNA World, and the archaeal system is apparently the most primative of these. Secondly, ribonuclease P is a great potential target for the development of new antimicrobials. Lastly, biophysicists generally, and most specifically in the field of RNA, have flocked to archaeal systems because of their simplicity and the relative ease of biophysical analysis of the RNAs, proteins, and RNA:protein complexes from these organisms.
Publications
- Brown, J.W. and Ellis, J.C. 2005 Comparative analysis of RNA secondary structure: The 6S RNA. In Handbook of RNA Biochemistry (Wiley-VCH), A. Bindereif, R. Hartmann, A. Schoen, and E. Westhof, eds.
- Marquez, S.M., Harris, J.K, Kelley, S.T., Brown, J.W., Dawson, S.C., Roberts, E.C. and Pace, N.R. 2005 Structural implications of novel diversity in eucaral RNase P RNA. RNA 11:739-751
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Progress 10/01/03 to 09/30/04
Outputs
The first goal of this project is to identify all of he subunits of RNase P in M.thermoautotrophicus, in specific to test whether or not Mth1483p is a subunit of RNase P, and to determine the structures of theese proteins (presumably in collaboration). Toward this goal, we have determined that Mth1483p is not physically associated with RNase P nor is it required (or beneficial) for enzymatic activity in vitro; i.e. it is apparently not an RNase P subunit. The assocation of the homolog of this protein in humans with RNase P, then, seems to be idiosynchratic to humans (and presumably animals generally). This has focused attention on the four core proteins, which have now been shown to be necessary and sufficient (along with the catalytic RNA, of course) for activity. The second goal is to determine the interactions between subunits, and how they contribute towards function. We have identified the protein:protein interactions using the yeast 2-hybrid system, and will
work to identify the protein:RNA interactions. Toward goal three (similar questions on the more complex type M enzyme from M.jannaschii), we have identified a number of potential RNase P protein subunits in M.jannaschii, and are currently examining their association with the RNA and RNase P activity.
Impacts
Our recent work on RNase P in Archaea has strongly focused interest in the homologs of the 4 archaeal proteins of the very complex eukaryotic nuclear RNase P; in fact it has been reported that the human RNase P RNA can be rendered functional with only these proteins (the native form contains at least an additional 8 proteins), although this claim has proven difficult to substantiate. Several structural biochemists are working to solve the structures of the archaeal proteins, and some have been determined; interestingly, none of these adopt a fold related to the bacterial protein.
Publications
- Williams, D. and Brown, J.W. 2004 Artificial and evolutionary variation in an archaeal RNase P RNA. Archaea 1:241-245.
- Ellis, J.C., and Brown, J.W. 2003 Genes with genes within Bacteria. Trends Biochem. Sci. 28:521-523.
- Tran, E., Brown, J.W., and Maxwell, E.S. 2004 Evolutionary origins of the RNA-guided nucleotide modification complexes: From the primitive translational apparatus? Trends Biochem. Sci. 29:343-350.
- Hall, T.A. and Brown, J.W. 2004 Protein-protein interactions between archaeal RNase P protein subunits. Archaea 1:247-253.
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