Dynamics of DNA-Bending in Binding Site Recognition by IHF
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HMG-box
DNA binding site
DNA binding site
Inverted repeat
A-site
HMG-box
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The c-Myb oncoprotein is known to bind to multiple sites in the promoters of target genes. We have developed a protocol to screen the binding site of c-Myb by using the systematic binding data derived from measurements of binding affinity for oligonucleotide containing a known Myb-binding site and its complete single mutants. We first applied the method to predict the binding affinity for the known binding sites and compared with available experimental data. The predicted binding sites agree with many putative binding sites of known target promoters. However, there are some binding sites not predicted by the analysis. These sequences deviate from the consensus sequence derived from the binding analyses. In the light of the structure of Myb-DNA complex, these results indicate that different DNA-binding modes may be used by c-Myb to recognize different classes of binding sites. We also screened the sequence database for potential Myb-binding sites, and found sequences of several promoters that have not been identified experimentally but could be the target for c-Myb.
MYB
DNA binding site
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Gene expression is controlled primarily by transcription factors, whose DNA binding sites are typically 10 nt long. We develop a population-genetic model to understand how the length and information content of such binding sites evolve. Our analysis is based on an inherent trade-off between specificity, which is greater in long binding sites, and robustness to mutation, which is greater in short binding sites. The evolutionary stable distribution of binding site lengths predicted by the model agrees with the empirical distribution (5-31 nt, with mean 9.9 nt for eukaryotes), and it is remarkably robust to variation in the underlying parameters of population size, mutation rate, number of transcription factor targets, and strength of selection for proper binding and selection against improper binding. In a systematic data set of eukaryotic and prokaryotic transcription factors we also uncover strong relationships between the length of a binding site and its information content per nucleotide, as well as between the number of targets a transcription factor regulates and the information content in its binding sites. Our analysis explains these features as well as the remarkable conservation of binding site characteristics across diverse taxa.
DNA binding site
Transcription
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We have used the technique known as selected and amplified binding (SAAB) to Isolate binding sites for the yeast transcription factor RAP1 from a degenerate pool of oligonucleotldes. A total of 47 sequences were isolated, of which two were shown to be contaminating non-RAP1 binding sites. After excluding these two sequences the remainder of the sequences were used to derive a new consensus binding site for RAP1. The new consensus 5′ A/G T A/G C A C C C A N N C C/A C C 3′ is a significant extension of the existing consensus (4). It is longer by two base pairs at the 5′ end and is significantly more constrained at the 3′ end. An analysis of the combinations of mis-matches In individual SAAB sequences, compared to the consensus RAP1 binding site, has allowed us to analyse the structure of the RAP1 binding site In some detail. The binding site can be sub-divided into three regions; a core binding site, a 5′ flanking region and a 3′ flanking region. The core binding site, consisting of the sequence 5CACCCA3′, is critical for recognition by RAP1. The less conserved flanking regions are not as Important. Interactions between RAP1 and these regions probably stabilise the Interaction between RAP1 and the core binding site. Each of the sequences isolated in the SAAB analysis was used to search release 78 of the EMBL + GenBank DNA data base. The searches Identified 102 potential binding sites for RAP1 within promoters of yeast genes.
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Rap1
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The fushi tarazu (ftz) gene of Drosophila melanogaster encodes a homeodomain-containing transcription factor that functions in the formation of body segments. Here we report an analysis of the DNA-binding properties of the ftz homeodomain in vitro. We provide evidence that the homeodomain binds to DNA as a monomer, with an equilibrium dissociation constant of 2.5 x 10(-11) M for binding to a consensus binding site. A single ftz binding site occupies 10 to 12 bp, as judged by the ability of protein bound at one site to interfere with binding to an adjacent site. These experiments also demonstrated a lack of cooperative binding between ftz homeodomains. Analysis of single-nucleotide substitutions over an 11-bp sequence shows that a stretch of 6 bp is critical for binding, with an optimal sequence of 5'CTAATTA3'. These data correlate well with recent structural evidence for base-specific contact at these positions. In addition, we found that sequences flanking the region of direct contact have effects on DNA binding that could be of biological significance.
DNA binding site
Dissociation constant
Cooperative binding
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bZIP domain
DNA binding site
HMG-box
Cytosine
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In animals, transcription factor binding sites are hard to recognize because of their extensive variation. We therefore characterized the general relationship between a specific protein‐binding site and its DNA sequence and used this relationship to generate a predictive algorithm for searching other DNA sequences. The experimental process was defined by studying hepatocyte nuclear factor 1 (HNF1), which binds DNA as a dimer on two inverted‐repeat 7‐bp half sites separated by one base. The binding model was based on the equivalence of the two half sites, which was confirmed in examples where specific modified sites were compared. Binding competition analysis was used to determine the effects of substitution of all four bases at each position in the half site. From these data, a weighted half‐site matrix was generated and the full site was evaluated as the sum of two half‐site scores. This process accurately predicted even weak binding sites that were significantly different from the consensus sequence. The predictions also showed a direct correlation with measured protein binding.
DNA binding site
Hepatocyte nuclear factors
A-site
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DNA binding site
HMG-box
Single-stranded binding protein
Linker
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DNA binding site
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DNA binding site
HMG-box
B3 domain
bZIP domain
Sequence motif
Protein–DNA interaction
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