Book of Abstracts: Albany 2003

category image Albany 2003
Conversation 13
Abstract Book
June 17-21 2003

Interaction of Plasmid pSM19035-encoded Regulatory Protein Omega with Operator DNA

Homodimeric ω protein (ω2) regulates transcription of genes required for control of plasmid copy number and stable inheritance (1). ω2 (71 amino acids per chain, 16 kDa) belongs to the MetJ/Arc structural superfamily of repressors forming a ribbon-helix-helix DNA binding motif (2, 3), and binds specifically promoter regions containing multiple consecutive copies of heptad sequences 5?- A/TATCACA/T-3? (top strand).

At least two consecutive copies of the 7-bp consensus repeat are necessary for high affinity binding of ω2, as shown by isothermal titration calorimetry and thermal unfolding experiments monitored by circular dichroism. We studied oligonucleotides composed of two heptads in three different orientations (head-to-head; head-to-tail; tail-to-tail¸ e.g. top strand 5?-GCG AATCACA TGTGATT GG-3?) and flanked by 3 and 2 bp at the 5?- and 3?-ends. Fluorescence titration experiments demonstrated the binding of one ω2 to one heptad.

Large effects of complex formation on protein and DNA were suggested by Raman measurements (4) pointing to an induced fit of the structures. We measured spectra of ω2 protein, oligonucleotides and ω2:oligonucleotide complexes as shown for instance in the figure for the head-to-head diheptad.

The difference spectrum (C) obtained by subtraction of the sum of the component spectra (A) from the spectrum of the complex (B) reveals significant perturbations inferring remarkable structural changes upon complex formation, e.g. conformational changes of the sugar-phosphate backbones, purine unstacking and altered furanose geometry (DNA bending) in the DNA, but also large changes in the protein. Surprisingly, similar effects were observed for all three orientations of the heptads suggesting a high adaptability of the components.

L. Dostál1
R. Misselwitz1
K. Welfle1
W. Saenger2
J. C. Alonso3
H. Welfle1,*

1Max-Delbrueck-Centrum fuer Molekulare Medizin Berlin-Buch
Robert-Roessle-Str. 10
D-13092 Berlin
2Institut fuer Kristallographie
Freie Universitaet Berlin
D-14195 Berlin, Germany
3Departmento de Biotecnología Microbiana
Centro Nacional de Biotecnología
E-28049, Madrid, Spain

References and Footnotes
  1. de la Hoz, A. B., Ayora, S., Sitkiewicz, I., Fernandez, S., Pankiewicz, R., Alonso, J. C. and Ceglowski, P., Proc. Natl. Acad. Sci. USA 97, 728-733 (2000).
  2. Misselwitz, R., de la Hoz, A. B., Ayora, S., Welfle, K., Behlke, J., Murayama, K., Saenger, W., Alonso, J. C., and Welfle, H., FEBS Lett. 505, 436-440 (2001).
  3. Murayama, K., Orth, P., de la Hoz, A. B., Alonso, J. C., and Saenger, W., J. Mol. Biol. 314, 789-796 (2001).
  4. Dostál, L., Misselwitz, R., Laettig, S., Alonso, J. C., Welfle, H., Spectroscopy, in press (2003).