Quantitative modeling of DNA-protein interactions: effects of amino acid substitutions on binding specificity of the Mnt repressor.
Understanding DNA-protein recognition quantitatively is essential to developing computational algorithms for accurate transcriptional binding site prediction. Using a quantitative, multiple fluorescence, relative affinity (QuMFRA) assay, we determine the binding specificity of 11 different position 6 variants of the Mnt repressor for operators containing all 16 possible dinucleotides at operator positions 16 and 17. We show that the wild-type and all variant proteins interact with the two positions in a non-independent manner, but that a simple independent model provides a close approximation to the true binding affinities. The wild-type His at amino acid 6 is the only protein to prefer the AC sequence of the wild-type operator, whereas most of the variant proteins prefer TA. H6R is unique in having a strong preference for C at position 16. A comparison of the quantitative binding data for all of the protein variants with a model for recognition of the early growth response (EGR) zinc finger family suggests that interactions of Mnt with positions 16 and 17 are similar to interactions of EGR with positions 1 and 2, respectively. This information leads to an augmented model for the interaction of Mnt with its operator.
Man
Tsz-Kwong
TK
Department of Genetics, Washington University, 660 S. Euclid, Box 8232, St Louis, MO 63110, USA.
Yang
Joshua SungWoo
JS
Stormo
Gary D
GD
D019943:Amino Acid Substitution; D001665:Binding Sites; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008957:Models, Genetic; D009875:Operator Regions, Genetic; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D054334:Viral Regulatory and Accessory Proteins
D004268:DNA-Binding Proteins; D012097:Repressor Proteins; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; C016649:phage repressor proteins; D004247:DNA