T7 RNA Polymerase in a High Magnetic Field

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

18.9 T
Control
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/winter200
5/stryer/ch28/Slide9.jpg
5
T7 RNA PolymeraseThe Hand Model
Structure found in protein data base PDB 1QLN
(Tahirov et al. 2002). Thumb wraps around DNA
(Gopal 1999)
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
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

Lu 1999
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
  Thumbs 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.