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Engineering transcription factors with novel DNA-binding specificity using comparative genomics.

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  • Author(s): Desai TA;Desai TA; Rodionov DA; Gelfand MS; Alm EJ; Rao CV
  • Source:
    Nucleic acids research [Nucleic Acids Res] 2009 May; Vol. 37 (8), pp. 2493-503. Date of Electronic Publication: 2009 Mar 05.
  • Publication Type:
    Journal Article; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, Non-P.H.S.
  • Language:
    English
  • Additional Information
    • Source:
      Publisher: Oxford University Press Country of Publication: England NLM ID: 0411011 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1362-4962 (Electronic) Linking ISSN: 03051048 NLM ISO Abbreviation: Nucleic Acids Res Subsets: MEDLINE
    • Publication Information:
      Publication: 1992- : Oxford : Oxford University Press
      Original Publication: London, Information Retrieval ltd.
    • Subject Terms:
      Genomics*; DNA-Binding Proteins/*genetics ; Protein Engineering/*methods ; Transcription Factors/*genetics; Amino Acid Substitution ; Cyclic AMP Receptor Protein/chemistry ; Cyclic AMP Receptor Protein/genetics ; Cyclic AMP Receptor Protein/metabolism ; DNA-Binding Proteins/chemistry ; DNA-Binding Proteins/metabolism ; Escherichia coli Proteins/chemistry ; Escherichia coli Proteins/genetics ; Escherichia coli Proteins/metabolism ; Genes, Reporter ; Iron-Sulfur Proteins/chemistry ; Iron-Sulfur Proteins/genetics ; Iron-Sulfur Proteins/metabolism ; Mutation ; Operator Regions, Genetic ; Transcription Factors/chemistry ; Transcription Factors/metabolism ; Transcription, Genetic
    • Abstract:
      The transcriptional program for a gene consists of the promoter necessary for recruiting RNA polymerase along with neighboring operator sites that bind different activators and repressors. From a synthetic biology perspective, if the DNA-binding specificity of these proteins can be changed, then they can be used to reprogram gene expression in cells. While many experimental methods exist for generating such specificity-altering mutations, few computational approaches are available, particularly in the case of bacterial transcription factors. In a previously published computational study of nitrogen oxide metabolism in bacteria, a small number of amino-acid residues were found to determine the specificity within the CRP (cAMP receptor protein)/FNR (fumarate and nitrate reductase regulatory protein) family of transcription factors. By analyzing how these amino acids vary in different regulators, a simple relationship between the identity of these residues and their target DNA-binding sequence was constructed. In this article, we experimentally tested whether this relationship could be used to engineer novel DNA-protein interactions. Using Escherichia coli CRP as a template, we tested eight designs based on this relationship and found that four worked as predicted. Collectively, these results in this work demonstrate that comparative genomics can inform the design of bacterial transcription factors.
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    • Grant Information:
      55005610 United States Howard Hughes Medical Institute
    • Accession Number:
      0 (Cyclic AMP Receptor Protein)
      0 (DNA-Binding Proteins)
      0 (Escherichia coli Proteins)
      0 (FNR protein, E coli)
      0 (Iron-Sulfur Proteins)
      0 (Transcription Factors)
      0 (crp protein, E coli)
    • Publication Date:
      Date Created: 20090307 Date Completed: 20090624 Latest Revision: 20211020
    • Publication Date:
      20240513
    • Accession Number:
      PMC2677863
    • Accession Number:
      10.1093/nar/gkp079
    • Accession Number:
      19264798