Karin Musier-Forsyth
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Professor and Ohio Eminent Scholar
in Biological Macromolecular Structure
(joint appointment with the Department of Chemistry)
Phone: 614-292-2021
Fax: 614-688-5402
email: musier@chemistry.ohio-state.edu
Webpage: Musier-Forsyth Homepage |
Research Interests:
My group uses a wide variety of chemical and biophysical approaches to answer fundamental questions focusing on nucleic acids (RNA and DNA) and proteins that are involved in translation of the genetic code and viral replication.
Aminoacyl-tRNA Synthetases. Members of this ancient family of proteins catalyze the esterification of an amino acid onto the 3'-end of tRNAs, which translate the genetic code through codon-anticodon recognition. An evolutionary conserved 5¡Ç-monophosphate that is critical for efficient aminoacylation by E. coli and S. cerevisiae HisRS. By virtue of their specific recognition of tRNAs and their capability to discriminate among structurally similar amino acids, the aminoacyl-tRNA synthetases play a central role in assuring a high degree of accuracy in protein synthesis. Site-directed and atomic group "mutagenesis" are common methods used in the lab to probe tRNA recognition. The latter approach allows us to identify specific functional groups that contribute to synthetase recognition of RNA substrates. Overuse of antibiotics in combination with increased numbers of immunocompromised patients has led to a dramatic increase in the incidence of antibiotic resistance. The aminoacyl-tRNA synthetases are an essential family of enzymes offering up to twenty targets per pathogen. With this in mind, we are probing species-specific differences in substrate recognition for drug design and to gain insights into the development of aminoacylation systems through evolution. In comparison to our understanding of tRNA discrimination, relatively little is known about the mechanism synthetases use to discriminate amongst structurally related amino acids. To correct misactivation of noncognate substrates, many synthetases require editing activity, and studies aimed at understanding the mechanism of translational editing are also underway. A novel triple-sieve mechanism for ProRS editing that has been proposed by our lab. One goal of this work is to use our knowledge of amino acid recognition and editing to engineer novel synthetase-tRNA pairs for the incorporation of unnatural amino acids into proteins in vitro and in vivo.
Nucleic Acid-Protein Interactions in HIV. During the life cycle of HIV, its RNA genome must be converted into DNA. This conversion is catalyzed by reverse transcriptase, an enzyme that uses a specific host cell tRNALys molecule as a primer. The process by which HIV selects and uses a specific primer tRNA is not well understood, but we have recently shown that human lysyl-tRNA synthetase (LysRS) is also packaged into HIV and appears to be a critical factor in specific tRNA packaging. Ongoing work is aimed at elucidating the molecular interactions between human LysRS and HIV proteins. In vivo, the tRNA primer and the HIV RNA genome must be unwound and annealed together before reverse transcription can be initiated. The annealing process is mediated by the HIV nucleocapsid protein (NC), a nucleic acid "chaperone" protein that facilitates nucleic acid rearrangements. We have reconstituted in vitro systems that closely mimic several steps of the reverse transcription process in HIV to elucidate the mechanism of NCs chaperone funtion. Experimental approaches we are currently using to elucidate key nucleic acid-protein interactions in this system include fluorescence resonance energy transfer (FRET), chemical footprinting, and single molecule DNA stretching.
Publications
Selected publications from the last 3 years:
Cruceanu M, Gorelick RJ, Musier-Forsyth K, Rouzina I, Williams MC. (2006) "Rapid kinetics of protein-nucleic acid interaction is a major component of HIV-1 nucleocapsid protein's nucleic acid chaperone function." J Mol Biol. 363(5), 867-77.
Vo MN, Barany G, Rouzina I, Musier-Forsyth K. (2006) "Mechanistic studies of mini-TAR RNA/DNA annealing in the absence and presence of HIV-1 nucleocapsid protein." J Mol Biol. 363(1), 244-61.
Hati S, Ziervogel B, Sternjohn J, Wong FC, Nagan MC, Rosen AE, Siliciano PG, Chihade JW, Musier-Forsyth K. (2006) "Pre-transfer editing by class II prolyl-tRNA synthetase: role of aminoacylation active site in "selective release" of noncognate amino acids." J Biol Chem. 281(38), 27862-72.
Rosen AE, Brooks BS, Guth E, Francklyn CS, Musier-Forsyth K. (2006) "Evolutionary conservation of a functionally important backbone phosphate group critical for aminoacylation of histidine tRNAs." RNA. 12(7), 1315-22.
Kovaleski BJ, Kennedy R, Hong MK, Datta SA, Kleiman L, Rein A, Musier-Forsyth K. (2006) "In vitro characterization of the interaction between HIV-1 Gag and human lysyl-tRNA synthetase." J Biol Chem. 281(28), 19449-56.
Cosa G, Zeng Y, Liu HW, Landes CF, Makarov DE, Musier-Forsyth K, Barbara PF. (2006) "Evidence for non-two-state kinetics in the nucleocapsid protein chaperoned opening of DNA hairpins." J Phys Chem B Condens Matter Mater Surf Interfaces Biophys." 110(5), 2419-26.
Pinto YY, Le JD, Seeman NC, Musier-Forsyth K, Taton TA, Kiehl RA. (2005) "Sequence-encoded self-assembly of multiple-nanocomponent arrays by 2D DNA scaffolding." Nano Lett. 5(12), 2399-402.
Levin JG, Guo J, Rouzina I, Musier-Forsyth K. (2005) "Nucleic acid chaperone activity of HIV-1 nucleocapsid protein: Critical role in reverse transcription and molecular mechanism." Prog Nucleic Acid Res Mol Biol. 80, 217-86. Review.
Liu HW, Cosa G, Landes CF, Zeng Y, Kovaleski BJ, Mullen DG, Barany G, Musier-Forsyth K, Barbara PF. (2005) "Single-molecule FRET studies of important intermediates in the nucleocapsid-protein-chaperoned minus-strand transfer step in HIV-1 reverse transcription." Biophys J. 89(5), 3470-9.
An S, Musier-Forsyth K. (2005) "Cys-tRNA(Pro) editing by Haemophilus influenzae YbaK via a novel synthetase.YbaK.tRNA ternary complex." J Biol Chem. 280(41), 34465-72.
Kankia BI, Barany G, Musier-Forsyth K. (2005) "Unfolding of DNA quadruplexes induced by HIV-1 nucleocapsid protein." Nucleic Acids Res. 33(14), 4395-403.
Cosa G, Harbron EJ, Zeng Y, Liu HW, O'Connor DB, Eta-Hosokawa C, Musier-Forsyth K, Barbara PF. (2004) "Secondary structure and secondary structure dynamics of DNA hairpins complexed with HIV-1 NC protein." Biophys J. 87(4), 2759-67.
An S, Musier-Forsyth K. (2004) "Trans-editing of Cys-tRNAPro by Haemophilus influenzae YbaK protein." J Biol Chem. 279(41), 42359-62.
Gunasekera N, Lee SW, Kim S, Musier-Forsyth K, Arriaga E. (2004) "Nuclear localization of aminoacyl-tRNA synthetases using single-cell capillary electrophoresis laser-induced fluorescence analysis." Anal Chem. 76(16), 4741-6.
Gunasekera N, Xiong G, Musier-Forsyth K, Arriaga E. (2004) "A capillary electrophoretic method for monitoring the presence of alpha-tubulin in nuclear preparations." Anal Biochem. 330(1), 1-9.
Hargittai MR, Gorelick RJ, Rouzina I, Musier-Forsyth K. (2004) "Mechanistic insights into the kinetics of HIV-1 nucleocapsid protein-facilitated tRNA annealing to the primer binding site." J Mol Biol. 337(4), 951-68.
Gunasekera N, Olson KJ, Musier-Forsyth K, Arriaga EA. (2004) "Capillary electrophoretic separation of nuclei released from single cells." Anal Chem. 76(3), 655-62.
Connolly SA, Rosen AE, Musier-Forsyth K, Francklyn CS. (2004) "G-1:C73 recognition by an arginine cluster in the active site of Escherichia coli histidyl-tRNA synthetase." Biochemistry. 43(4), 962-9.
Rosen AE, Musier-Forsyth K. (2004) "Recognition of G-1:C73 atomic groups by Escherichia coli histidyl-tRNA synthetase." J Am Chem Soc. 126(1), 64-5.
Seetharaman M, Williams C, Cramer CJ, Musier-Forsyth K. (2003) "Effect of G-1 on histidine tRNA microhelix conformation." Nucleic Acids Res. 31(24), 7311-21.
Wong FC, Beuning PJ, Silvers C, Musier-Forsyth K. (2003) "An isolated class II aminoacyl-tRNA synthetase insertion domain is functional in amino acid editing." J Biol Chem. 278(52), 52857-64.
Lee N, Gorelick RJ, Musier-Forsyth K. (2003) "Zinc finger-dependent HIV-1 nucleocapsid protein-TAR RNA interactions." Nucleic Acids Res. 31(16), 4847-55.
Khandogin J, Musier-Forsyth K, York DM. (2003) "Insights into the regioselectivity and RNA-binding affinity of HIV-1 nucleocapsid protein from linear-scaling quantum methods." J Mol Biol. 330(5), 993-1004.
Javanbakht H, Halwani R, Cen S, Saadatmand J, Musier-Forsyth K, Gottlinger H, Kleiman L. (2003) "The interaction between HIV-1 Gag and human lysyl-tRNA synthetase during viral assembly." J Biol Chem. 278(30), 27644-51.
Iwatani Y, Rosen AE, Guo J, Musier-Forsyth K, Levin JG. (2003) "Efficient initiation of HIV-1 reverse transcription in vitro. Requirement for RNA sequences downstream of the primer binding site abrogated by nucleocapsid protein-dependent primer-template interactions." J Biol Chem. 278(16), 14185-95.
Hong MK, Harbron EJ, O'Connor DB, Guo J, Barbara PF, Levin JG, Musier-Forsyth K. (2003) "Nucleic acid conformational changes essential for HIV-1 nucleocapsid protein-mediated inhibition of self-priming in minus-strand transfer." J Mol Biol. 325(1), 1-10.
