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Iterative Optimization of DNA Duplexes for Crystallization of SeqA-DNA Complexes
Published on: November 1, 2012
Crystallization and preliminary X-ray analysis of phage Mu activator protein C in a complex with promoter DNA
Karthik K Shanmuganatham1, Manimekalai Ravichandran, Martha M Howe
1Department of Molecular Sciences, University of Tennessee Health Science Center, Memphis, TN 38163, USA.
Abstract:
Bacteriophage Mu C protein is an activator of the four Mu late promoters that drive the expression of genes encoding DNA-modification as well as phage head and tail morphogenesis proteins. This report describes the purification and cocrystallization of wild-type and selenomethionine-substituted C protein with a synthetic late promoter P(sym), together with preliminary X-ray diffraction data analysis using SAD phasing. The selenomethionine peak data set was collected from a single crystal which diffracted to 3.1 A resolution and belonged to space group P4(1) or P4(3), with unit-cell parameters a = 68.9, c = 187.6 A and two complexes per asymmetric unit. The structure will reveal the amino acid-DNA interactions and any conformational changes associated with DNA binding.
Insights
Bacteriophage Mu C protein, a key activator, was purified and crystallized with a synthetic promoter. This structural study using X-ray diffraction aims to reveal DNA-binding interactions for bacteriophage Mu.
Area of Science:
- Molecular Biology
- Structural Biology
- Virology
Background:
- Bacteriophage Mu C protein activates late promoters essential for phage gene expression.
- These promoters control genes involved in DNA modification and phage structure formation.
Purpose of the Study:
- To determine the structure of Bacteriophage Mu C protein bound to a synthetic late promoter.
- To elucidate the molecular mechanisms of DNA binding and transcriptional activation by C protein.
Main Methods:
- Purification of wild-type and selenomethionine-substituted C protein.
- Cocrystallization of C protein with a synthetic promoter DNA (P(sym)).
- X-ray diffraction data collection and preliminary analysis using Single-wavelength Anomalous Dispersion (SAD) phasing.
Main Results:
- A single crystal diffracted to 3.1 Å resolution.
- The crystal belonged to space group P4(1) or P4(3) with specific unit-cell parameters.
- Two complexes were found per asymmetric unit, indicating potential dimeric or multimeric interactions.
Conclusions:
- The determined structure will provide insights into amino acid-DNA interactions.
- Conformational changes upon DNA binding will be revealed.
- This structural information is crucial for understanding bacteriophage Mu gene regulation.
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