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Solution studies on the structure of bent DNA in the cAMP receptor protein-lac DNA complex
1Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston 77550.
Abstract:
Cyclic AMP receptor protein is involved in the regulation of more than 20 genes. A step in the mechanism of activation of transcription is to induce a significant bending of the DNA upon complex formation between specific DNA and the protein. The induced DNA bending and a structure of the protein-DNA complex were studied by fluorescence energy transfer in 50 mM Tris, 1 mM EDTA, and 50 mM KCl at pH 7.8 and 20 degrees C. The symmetry of the DNA bend was estimated by measuring the efficiency of transfer between the protein and a label on either the upstream or the downstream end of a lac DNA fragment. The results show that the bend, despite the asymmetry in the DNA sequence, is symmetrical, for the fragments which length ranges from 26 to 40 bp. Using fluorescence energy transfer, the extent of DNA bending was estimated by measuring the end-to-end distance of the DNA fragment which was labeled with a donor-acceptor pair on two opposite ends. Both steady-state and time-resolved measurements showed that in a 26 bp lac DNA fragment complexed with cyclic AMP receptor protein, the end-to-end distance is about 77 A which corresponds to a bending angle of 80 degrees or 100 degrees, depending on the actual contour length between the fluorophores in the free DNA fragment. The results using longer DNA fragments show no measurable amount of energy transfer; thus, it is very unlikely that the DNA completely wraps around the CRP molecule.(ABSTRACT TRUNCATED AT 250 WORDS)
Insights
Cyclic AMP receptor protein induces a symmetrical DNA bend upon binding, with a 26 bp lac DNA fragment bending 80-100 degrees. This DNA bending is crucial for gene regulation.
Area of Science:
- Molecular Biology
- Biophysics
Background:
- Cyclic AMP receptor protein (CRP) regulates over 20 genes.
- CRP-DNA complex formation induces significant DNA bending, a key step in transcription activation.
Purpose of the Study:
- To investigate the DNA bending induced by CRP.
- To determine the structure of the CRP-DNA complex and the symmetry of DNA bending.
Main Methods:
- Fluorescence energy transfer (FRET) was used to study DNA bending and protein-DNA complex structure.
- Measurements were conducted in Tris buffer (50 mM, pH 7.8) with 1 mM EDTA and 50 mM KCl at 20°C.
- FRET efficiency was measured using labeled lac DNA fragments (26-40 bp) and CRP.
Main Results:
- The DNA bend induced by CRP is symmetrical, even with asymmetric DNA sequences, for fragments 26-40 bp long.
- A 26 bp lac DNA fragment complexed with CRP showed an end-to-end distance of ~77 Å, corresponding to a bending angle of 80-100 degrees.
- No significant energy transfer was observed with longer DNA fragments, suggesting DNA does not fully wrap around CRP.
Conclusions:
- CRP binding induces a symmetrical DNA bend of approximately 80-100 degrees in short DNA fragments.
- The study provides insights into the structural mechanism of CRP-mediated transcription regulation.