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T7 RNA Polymerase in a High Magnetic Field

T7 RNA Polymerase in a High Magnetic Field. Kim Wadelton Sweet Briar College. Sponsored by: The National High Magnetic Field Laboratory The University of Florida The National Science Foundation (NSFDMR-0305371) and NASA (NNA045561). Marianna Worczak Clarkson University James Ch. Davis

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T7 RNA Polymerase in a High Magnetic Field

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  1. T7 RNA Polymerase in a High Magnetic Field Kim Wadelton Sweet Briar College Sponsored by: The National High Magnetic Field Laboratory The University of Florida The National Science Foundation (NSFDMR-0305371) and NASA (NNA045561) Marianna Worczak Clarkson University James Ch. Davis University of Florida Anna-Lisa Paul University of Florida Department of Horticultural Sciences Mark W. Meisel University of Florida Department of Physics and NHMFL

  2. Background • NASA experimented with High Magnetic Fields to simulate zero gravity for experiments on plants • Paul and coworkers found that the magnetic field itself, not just the lack of gravity, was causing stress on the plants Control 18.9 T Paul 2005

  3. Hypothesis • Strong magnetic fields generate subtle perturbations of biomolecules due to the structural diamagnetic anisotropy of the molecules, causing a disruption of normal biochemical function m B

  4. Transcription RNA Polymerase RNA Transcript http://oregonstate.edu/instruction/bb451/winter2005/stryer/ch28/Slide9.jpg

  5. T7 RNA Polymerase:The Hand Model Structure found in protein data base PDB# 1QLN (Tahirov et al. 2002).

  6. Top View Structure found in protein data base PDB# 1QLN (Tahirov et al. 2002).

  7. 1D Model – No Field Fingers DNA Thumb 2 nm .5 nm .5 nm Palm Approximate dimensions given

  8. 1D Model – Field s = 1 nm Fingers FM DNA DNA Thumb 2 nm FR .5 nm Palm Approximate dimensions given

  9. Energy Analysis Compared to ambient Thermal Energy: *Worchester 1978 **Pauling 1979

  10. F F Force Analysis http://resumbrae.com/ub/dms424_s03/22/00.html

  11. Solve for Magnetic Force

  12. Is This Force Reasonable? • Other molecular forces creating extreme structural alterations: ~10-10-10-11 N • Overstretching DNA • Unfolding Titin (muscle) • Unfolding DNA hairpin • Preventing T7 RNAP from proceeding along the DNA during transcription

  13. Conclusions • Experimentally, • Some delay in transcript production is indicated at 9 Tesla for T7 RNA polymerase • A reduction in transcript production was observed for SP6 RNA polymerase at 9 Tesla • Theoretically, a more accurate model is needed • Improve k approximations • Improve force estimates • Include other possible deformations • Next: Further test the hypothesis • Analyze experiments at 20 and 25 Tesla

  14. T7 RNA Polymerase in a High Magnetic Field Kim Wadelton Sweet Briar College Sponsored by: The National High Magnetic Field Laboratory The University of Florida The National Science Foundation (NSFDMR-0305371) and NASA (NNA045561) Marianna Worczak Clarkson University James Ch. Davis University of Florida Anna-Lisa Paul University of Florida Department of Horticultural Sciences Mark W. Meisel University of Florida Department of Physics and NHMFL

  15. Force vs. Field Strength

  16. The Thumb • Large • Flexible* • Located at DNA entry pore* • Steadies DNA** Thumb * **

  17. References Cheetham, Graham M. T., David Jeruzalmi, and Thomas A Steitz (1999) Structural basis for initiation of transcription from an RNA polymerase-promoter complex. Nature (399) 80-83. Gopal, Vijaya et al (1999) Characterization of Structural Features Important for T7 RNAP Elongation Complex Stability Reveals Competing Complex Conformations and a Role for the Non-template strand in RNA Displacement. J. Mol. Biol. (290) 411-431 Lu, Hui and Klaus Schulten (1999) Steered Molecular Dynamics Simulations of Force-Induced Protein Domain Unfolding. PROTEINS: Structure, Function, and Genetics (35) 453-463 Paul, A.-L., R.J. Ferl, B. Klingenberg, J.S. Brooks, A.N. Morgan, J. Yowtak, and M.W. Meisel (2005) Strong Magnetic Field Induced Changes of Gene Expression in Arabidopsis. Materials Processing in Magnetic Fields: Proceedings of the International Workshop on Materials Analysis and Processes in Magnetic Fields (NHMFL, Tallahassee, 17-19 March 2004). To appear fall 2005. Pauling, Linus (1979) Diamagnetic anisotropy of the peptide group. Biophysics (76) 2293-2294. Sousa, Rui, John Rose and B. C. Wang (1994) The Thumb’s Knuckle: Flexibility in the Thumb Subdomain of T7 RNA Polymerase is Revealed by the Structure of a Chimeric T7/T3 RNA Polymerase.Jol. Mol. Biol. (244) 6-12. T7 RiboMax Express Large Scale RNA Production System Technical Bulletin. Promega. (www.promega.com) Tahirov, Tahir H et al. (2002) Structure of a T7 RNA polymerase elongation complex at 2.9 A resolution.Nature (420) 43-50. Wadelton, Kim et al. (2005) Diamagnetic Anisotropy of T7 RNA Polymerase Report of research preformed Summer 2005 as part of NHMFL REU Program. Worchester, D.L. (1978) Structural Origins of diamagnetic anisotropy. Pro.Natl. Acad. Sci. (75) 5475-5477. Worczak, Marianna et al. (2005) Effects of high magnetic fields on in vitro transcription Report of research preformed Summer 2005 as part of NHMFL REU Program.

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