Reengineering Cro protein functional specificity with an evolutionary code
| Autor: | Matthew H. J. Cordes, Adrian R. Begaye, Branwen M. Hall, Erin E. Vaughn |
|---|---|
| Rok vydání: | 2011 |
| Předmět: |
viruses
Helix-turn-helix Electrophoretic Mobility Shift Assay Fluorescence Polarization Computational biology DNA sequencing Nucleobase Substrate Specificity Bacteriophage Evolution Molecular chemistry.chemical_compound Structural Biology Two-Hybrid System Techniques Viral Regulatory and Accessory Proteins Molecular Biology Transcription factor Genetics chemistry.chemical_classification biology Circular Dichroism DNA biology.organism_classification Bacteriophage lambda Amino acid DNA-Binding Proteins Repressor Proteins chemistry Genetic Code Helix Genetic Engineering |
| Zdroj: | Journal of molecular biology. 413(5) |
| ISSN: | 1089-8638 |
| Popis: | Cro proteins from different lambdoid bacteriophages are extremely variable in their target consensus DNA sequences and constitute an excellent model for evolution of transcription factor specificity. We experimentally tested a bioinformatically derived evolutionary code relating switches between pairs of amino acids at three recognition helix sites in Cro proteins to switches between pairs of nucleotide bases in the cognate consensus DNA half-sites. We generated all eight possible code variants of bacteriophage λ Cro and used electrophoretic mobility shift assays to compare binding of each variant to its own putative cognate site and to the wild-type cognate site; we also tested the wild-type protein against all eight DNA sites. Each code variant showed stronger binding to its putative cognate site than to the wild-type site, except some variants containing proline at position 27; each also bound its cognate site better than wild-type Cro bound the same site. Most code variants, however, displayed poorer affinity and specificity than wild-type λ Cro. Fluorescence anisotropy assays on λ Cro and the triple code variant (PSQ) against the two cognate sites confirmed the switch in specificity and showed larger apparent effects on binding affinity and specificity. Bacterial one-hybrid assays of λ Cro and PSQ against libraries of sequences with a single randomized half-site showed the expected switches in specificity at two of three coded positions and no clear switches in specificity at noncoded positions. With a few caveats, these results confirm that the proposed Cro evolutionary code can be used to reengineer Cro specificity. |
| Databáze: | OpenAIRE |
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