Journal of Molecular Biology
CommunicationRationalization of Gene Regulation by a Eukaryotic Transcription Factor: Calculation of Regulatory Region Occupancy from Predicted Binding Affinities
Section snippets
Derivation of a Leu3 binding-site model
The DNA-binding domain of Leu3 (amino acid residues 1–147) was expressed and purified to near-homogeneity as a fusion protein using the pMAL system in Esherichia coli.3., 11., 12. An electrophoretic mobility shift assay (EMSA) was then used to determine the affinity of this protein for 50 variants of the binding site (Table 1).13 Of the 50 variants, 29 were obtained by an in vitro selection procedure.14., 15. The selection served as a way to obtain diverse variants that bind with moderate to
Calculation of occupancy and correlation with gene expression
Our purpose in deriving a model for binding affinity is to see how well that model can be used to rationalize gene regulation. In addition to a model for binding affinity, we need a model for how multiple binding sites contribute to gene regulation. As with the affinity model, the goal is to use models with as few parameters as possible and then add complexity to the model as necessary and when justified by the data. The simple regulatory model we have implemented here is that binding to any
Prospects for improvement
More elaborate models for binding affinity might improve our ability to rationalize gene expression. It has been suggested, for example, that dependencies between neighboring base-pairs could be determined systematically by measuring the binding constants for all 16 variants of a particular dinucleotide, and then representing the data in a 16×(N−1) weight matrix of overlapping dinucleotides.22 This weight matrix has many more parameters than the standard 4×N matrix of independent base-pairs and
Supplementary Files
References (24)
- et al.
Purification and structural characterization of transcriptional regulator Leu3 of yeast
J. Biol. Chem.
(1993) - et al.
Identification of regulatory regions which confer muscle-specific gene expression
J. Mol. Biol.
(1998) - et al.
Quantitative specificity of the Mnt repressor
J. Mol. Biol.
(1997) - et al.
Structure of yeast regulatory gene LEU3 and evidence that LEU3 itself is under general amino acid control
Nucl. Acids Res.
(1987) - et al.
The Saccharomyces cerevisiae Leu3 protein activates expression of GDH1, a key gene in nitrogen assimilation
Mol. Cell Biol.
(1995) - et al.
Transcriptional regulation of the Saccharomyces cerevisiae amino acid permease gene BAP2
Mol. Gen. Genet.
(2001) - et al.
LEU3 of Saccharomyces cerevisiae activates multiple genes for branched-chain amino acid biosynthesis by binding to a common decanucleotide core sequence
Mol. Cell Biol.
(1988) DNA binding sites: representation and discovery
Bioinformatics
(2000)- Clarke, N. D., Granek, J. A. (2002). Rank order metrics for quantifying the association of sequence features with gene...
- et al.
Genome-wide analysis of clustered Dorsal binding sites identifies putative target genes in the Drosophila embryo
Proc. Natl Acad. Sci. USA
(2002)