Limits...
Algorithm for prediction of tumour suppressor p53 affinity for binding sites in DNA.

Veprintsev DB, Fersht AR - Nucleic Acids Res. (2008)

Bottom Line: Based on measurements of the effects of every possible single base-pair substitution of a consensus sequence, we defined the DNA sequence with the highest affinity for full-length p53 and quantified the effects of deviation from it on the strength of protein-DNA interaction.But, in some cases we observed significant deviations from additivity.We used it to search for high-affinity binding sites in the genome and to predict the effects of single-nucleotide polymorphisms in these sites.

View Article: PubMed Central - PubMed

Affiliation: MRC Centre for Protein Engineering, Cambridge, CB2 0QH, UK. dbv@mrc-lmb.cam.ac.uk

ABSTRACT
The tumour suppressor p53 is a transcription factor that binds DNA in the vicinity of the genes it controls. The affinity of p53 for specific binding sites relative to other DNA sequences is an inherent driving force for specificity, all other things being equal. We measured the binding affinities of systematically mutated consensus p53 DNA-binding sequences using automated fluorescence anisotropy titrations. Based on measurements of the effects of every possible single base-pair substitution of a consensus sequence, we defined the DNA sequence with the highest affinity for full-length p53 and quantified the effects of deviation from it on the strength of protein-DNA interaction. The contributions of individual nucleotides were to a first approximation independent and additive. But, in some cases we observed significant deviations from additivity. Based on affinity data, we constructed a binding predictor that mirrored the existing p53 consensus sequence definition. We used it to search for high-affinity binding sites in the genome and to predict the effects of single-nucleotide polymorphisms in these sites. Although there was some correlation between the K(d) and biological function, the spread of the K(d)s by itself was not sufficient to explain the activation of different pathways by changes in p53 concentration alone.

Show MeSH

Related in: MedlinePlus

Quantitative sequence logo of the p53 DNA-binding preferences. The height of the bars represents the number of times by which the amount of protein bound decreases, due to the base substitution. It shows the biggest possible effect caused by three alternative substitutions. The height of individual letters is proportional to the amount of transcription factor bound to individual sequence variants under identical conditions. The sequence of the second half-site is identical to the first one when read on the non-coding strand of DNA in the 5′–3′ direction. The sequence logo takes complementarity of DNA into account. The sequence with the highest affinity for p53 is GG(A/G)CATGCCCGGGCATG(T/C)CC.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2275157&req=5

Figure 3: Quantitative sequence logo of the p53 DNA-binding preferences. The height of the bars represents the number of times by which the amount of protein bound decreases, due to the base substitution. It shows the biggest possible effect caused by three alternative substitutions. The height of individual letters is proportional to the amount of transcription factor bound to individual sequence variants under identical conditions. The sequence of the second half-site is identical to the first one when read on the non-coding strand of DNA in the 5′–3′ direction. The sequence logo takes complementarity of DNA into account. The sequence with the highest affinity for p53 is GG(A/G)CATGCCCGGGCATG(T/C)CC.

Mentions: The change of the affinity of interaction changes the amount of protein bound to this site as follows:1where [P] is the free concentration of the transcription factor and Kd is the affinity of interaction. While we do not know the exact value of [P] in the cell, the ChIP enrichment values are typically in the range of 1–10%, meaning that binding sites are only partially saturated in vivo. Therefore, we can assume that [P] < Kd. Under such conditions, the amount of protein bound would decrease as:2where n is the Hill constant of interaction which we have previously shown to be 2, for p53 (17). The importance of the co-operativity of interaction of the transcription factor with DNA cannot be stressed enough. It magnifies the effects of the change in the Kd on the relative amount of the protein–DNA complex formed. We converted the observed changes in the Kd to the sequence logo (18) (Figure 3).


Algorithm for prediction of tumour suppressor p53 affinity for binding sites in DNA.

Veprintsev DB, Fersht AR - Nucleic Acids Res. (2008)

Quantitative sequence logo of the p53 DNA-binding preferences. The height of the bars represents the number of times by which the amount of protein bound decreases, due to the base substitution. It shows the biggest possible effect caused by three alternative substitutions. The height of individual letters is proportional to the amount of transcription factor bound to individual sequence variants under identical conditions. The sequence of the second half-site is identical to the first one when read on the non-coding strand of DNA in the 5′–3′ direction. The sequence logo takes complementarity of DNA into account. The sequence with the highest affinity for p53 is GG(A/G)CATGCCCGGGCATG(T/C)CC.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2275157&req=5

Figure 3: Quantitative sequence logo of the p53 DNA-binding preferences. The height of the bars represents the number of times by which the amount of protein bound decreases, due to the base substitution. It shows the biggest possible effect caused by three alternative substitutions. The height of individual letters is proportional to the amount of transcription factor bound to individual sequence variants under identical conditions. The sequence of the second half-site is identical to the first one when read on the non-coding strand of DNA in the 5′–3′ direction. The sequence logo takes complementarity of DNA into account. The sequence with the highest affinity for p53 is GG(A/G)CATGCCCGGGCATG(T/C)CC.
Mentions: The change of the affinity of interaction changes the amount of protein bound to this site as follows:1where [P] is the free concentration of the transcription factor and Kd is the affinity of interaction. While we do not know the exact value of [P] in the cell, the ChIP enrichment values are typically in the range of 1–10%, meaning that binding sites are only partially saturated in vivo. Therefore, we can assume that [P] < Kd. Under such conditions, the amount of protein bound would decrease as:2where n is the Hill constant of interaction which we have previously shown to be 2, for p53 (17). The importance of the co-operativity of interaction of the transcription factor with DNA cannot be stressed enough. It magnifies the effects of the change in the Kd on the relative amount of the protein–DNA complex formed. We converted the observed changes in the Kd to the sequence logo (18) (Figure 3).

Bottom Line: Based on measurements of the effects of every possible single base-pair substitution of a consensus sequence, we defined the DNA sequence with the highest affinity for full-length p53 and quantified the effects of deviation from it on the strength of protein-DNA interaction.But, in some cases we observed significant deviations from additivity.We used it to search for high-affinity binding sites in the genome and to predict the effects of single-nucleotide polymorphisms in these sites.

View Article: PubMed Central - PubMed

Affiliation: MRC Centre for Protein Engineering, Cambridge, CB2 0QH, UK. dbv@mrc-lmb.cam.ac.uk

ABSTRACT
The tumour suppressor p53 is a transcription factor that binds DNA in the vicinity of the genes it controls. The affinity of p53 for specific binding sites relative to other DNA sequences is an inherent driving force for specificity, all other things being equal. We measured the binding affinities of systematically mutated consensus p53 DNA-binding sequences using automated fluorescence anisotropy titrations. Based on measurements of the effects of every possible single base-pair substitution of a consensus sequence, we defined the DNA sequence with the highest affinity for full-length p53 and quantified the effects of deviation from it on the strength of protein-DNA interaction. The contributions of individual nucleotides were to a first approximation independent and additive. But, in some cases we observed significant deviations from additivity. Based on affinity data, we constructed a binding predictor that mirrored the existing p53 consensus sequence definition. We used it to search for high-affinity binding sites in the genome and to predict the effects of single-nucleotide polymorphisms in these sites. Although there was some correlation between the K(d) and biological function, the spread of the K(d)s by itself was not sufficient to explain the activation of different pathways by changes in p53 concentration alone.

Show MeSH
Related in: MedlinePlus