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Non-independence of Mnt repressor-operator interaction determined by a new quantitative multiple fluorescence relative affinity (QuMFRA) assay
Open accessAbstract
Salmonella bacteriophage repressor Mnt belongs to the ribbon-helix-helix class of transcription factors. Previous SELEX results suggested that interactions of Mnt with positions 16 and 17 of the operator DNA are not independent. Using a newly developed high-throughput quantitative multiple fluorescence relative affinity (QuMFRA) assay, we directly quantified the relative equilibrium binding constants (K(ref)) of Mnt to operators carrying all the possible dinucleotide combinations at these two positions. Results show that Mnt prefers binding to C, instead of wild-type A, at position 16 when wild-type C at position 17 is changed to other bases. The measured K(ref) values of double mutants were also higher than the values predicted from single mutants, demonstrating the non-independence of these two positions. The ability to produce a large number of quantitative binding data simultaneously and the potential to scale up makes QuMFRA a valuable tool for the large-scale study of macromolecular interaction.
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Cited by
Mathelier, A., Fornes, O., Arenillas, D. J., Chen, C.-Y., Denay, G., Lee, J., Shi, W., Shyr, C., Tan, G., Worsley-Hunt, R., et al. (2016) · Nucleic Acids Research · doi:10.1093/nar/gkv1176 · Cited by 937
A systems approach to measuring the binding energy landscapes of transcription factors.
Maerkl, S. J., & Quake, S. R. (2007) · Science · doi:10.1126/science.1131007 · Cited by 584
Absence of a simple code: how transcription factors read the genome
Slattery, M., Zhou, T., Yang, L., Dantas Machado, A. C., Gordân, R., & Rohs, R. (2014) · Trends in Biochemical Sciences · doi:10.1016/j.tibs.2014.07.002 · Cited by 537
UniPROBE: an online database of protein binding microarray data on protein-DNA interactions
Newburger, D. E., & Bulyk, M. L. (2009) · Nucleic Acids Research · doi:10.1093/nar/gkn660 · Cited by 367
Kinney, J. B., Murugan, A., Callan, C. G., & Cox, E. C. (2010) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.1004290107 · Cited by 334
Metadata
- Authors
- Man, T.-K.
- Venue
- Nucleic Acids Research
- Publisher
- Oxford University Press (OUP)
- Published
- 2001-06-15
- Volume
- 29
- Issue
- 12
- Pages
- 2471-2478
- ISSN
- 0305-1048 (ISSN-L)
- 1362-4962
- OpenAlex
- W2071753700
- Cited by
- 189
- References
- 0
- FWCI
- 3.62
- Type
- journal article
- Language
- en
- Open access
- bronze
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- Man, T.-K. (2001). Non-independence of Mnt repressor-operator interaction determined by a new quantitative multiple fluorescence relative affinity (QuMFRA) assay. Nucleic Acids Research. https://doi.org/10.1093/nar/29.12.2471
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