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Hudson, B.P., Quispe, J., Lara-Gonzalez, S., Berman, H.M., Arnold, E., Ebright, R.H.,

EMD-5127 – PDB 3IYD Three-dimensional EM structure of an intact activator-dependent transcription initiation complex. Hudson, B.P., Quispe, J., Lara-Gonzalez, S., Berman, H.M., Arnold, E., Ebright, R.H., Lawson, C.L. (2009) Proc. National Acad. Sci. USA 106,   19830-19835. Acknowledgements.

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Hudson, B.P., Quispe, J., Lara-Gonzalez, S., Berman, H.M., Arnold, E., Ebright, R.H.,

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  1. EMD-5127 – PDB 3IYDThree-dimensional EM structure of an intact activator-dependent transcription initiation complex Hudson, B.P., Quispe, J., Lara-Gonzalez, S., Berman, H.M., Arnold, E., Ebright, R.H., Lawson, C.L. (2009) Proc. National Acad. Sci. USA 106,   19830-19835

  2. Acknowledgements http://roma.rutgers.edu Catherine Lawson, PI Helen Berman, co-PI Brian Benoff Jens Birktoft Brian Hudson ** Samuel Lara-Gonzalez Andrew Napoli Ramya Rao Kaitlyn Turo Collaborators Rutgers Richard Ebright Eddy Arnold Scripps Joel Quispe Bridget Carragher Clint Potter Funding: NIH GM021589

  3. Bacterial Transcription Activation 3 components: Activator (50 kDa) RNA polymerase (300 kDa) Promoter DNA (~100 base pairs) Total: ~ 500 kDa

  4. Sample Reconstitution +1 CAP (-61.5) -35 -10 ATAATCGCACCTTA ATAATCACACCTTA CGCAATAAATGTGATCTAGATCACATTTTAGGCAAAAAAGGCTTTACACTTTATGCTTCCGGCTCGT TGTGAGCGGATAACAAG GCGTTATTTACACTAGATCTAGTGTAAAATCCGTTTTTTCCGAAATGTGAAATACGAAGGCCGAGCA ACACTCGCCTATTGTTC

  5. EM Specimen • Carbon sandwich stain technique Carbon Film Uranyl Formate Stain Sample Carbon Film Grid Ohi et al, Biol. Proced. Online (2004), 6, 23-34.

  6. EM Reconstruction Particles (~17000 total) • EMAN projection matching • thermophilic RNAP starting model yielded density for E. coli-specificinsertions at expected positions First refinement cycle projections averages Final refinement cycle projections averages

  7. Resolution Estimation

  8. “Class I” Map + Interpretation EMDB ID EMD-5127 PDB ID 3iyd

  9. Initial Model Fitting T. thermophilus RNAP holoenzyme from Mukhopadhyay et al., 2008 (PDBid 3DXJ). CAP + CTD + DNA from Benoff et al., 2002 (PDBid 1LB2), CTD + 70 region 4 + DNA from Lara et al, manuscript in preparation DNA positions -23 to +20 from Lawson et al., 2004. E. coli subunit N-terminal domain dimer from Zhang & Darst, 1998 (PDBid 1BDF). E. coli70 regions 1.2 to 2 from Malhotra et al., 1996 (PDBid 1SIG). E. coli’GNCD from Chlenov et al., 2005 (PDBid 2AUK).

  10. Coordinate Model Generation • Rigid body fitting, multiple models (Chimera) • DNA helical path adjusted around -35 element (3DNA) • Unified RNAP homology model created (Modeller) • Domain-wise fitting (Yup.scx, improved overall map-model correlation coefficient from 0.77 to 0.85) • Geometry regularization (phenix.pdbtools)

  11. Coordinate Model Quality Phenix.pdbtools geometry regularization: • No close contacts • RMSD bonds 0.005 Å • RMSD angles 0.8 degrees • Unrealistic phi-psi angles: 500/4000 residues (12%)

  12. Reviewer Comment “the value of such a reconstruction is … in having a model for E. coli RNAP (which contains significant differences from the thermophilic enzymes) and in providing a framework for testing positions/interactions involving parts of the complex that have not been available previously. “

  13. Fitting Statistics

  14. Euler Frequency Distribution

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