The shape of the DNA minor groove directs binding by the DNA-bending protein Fis.

The bacterial nucleoid-associated protein Fis regulates diverse reactions by bending DNA and through DNA-dependent interactions with other control proteins and enzymes. In addition to dynamic nonspecific binding to DNA, Fis forms stable complexes with DNA segments that share little sequence conservation. Here we report the first crystal structures of Fis bound to high- and low-affinity 27-base-pair DNA sites. These 11 structures reveal that Fis selects targets primarily through indirect recognition mechanisms involving the shape of the minor groove and sequence-dependent induced fits over adjacent major groove interfaces. The DNA shows an overall curvature of approximately 65 degrees , and the unprecedented close spacing between helix-turn-helix motifs present in the apodimer is accommodated by severe compression of the central minor groove. In silico DNA structure models show that only the roll, twist, and slide parameters are sufficient to reproduce the changes in minor groove widths and recreate the curved Fis-bound DNA structure. Models based on naked DNA structures suggest that Fis initially selects DNA targets with intrinsically narrow minor grooves using the separation between helix-turn-helix motifs in the Fis dimer as a ruler. Then Fis further compresses the minor groove and bends the DNA to generate the bound structure.

[1]  M. Simon,et al.  Fis binding to the recombinational enhancer of the Hin DNA inversion system. , 1987, Genes & development.

[2]  S. McLeod,et al.  Mechanism of chromosome compaction and looping by the Escherichia coli nucleoid protein Fis. , 2006, Journal of molecular biology.

[3]  Dietrich Suck,et al.  Structure of DNase I at 2.0 Å resolution suggests a mechanism for binding to and cutting DNA , 1986, Nature.

[4]  H M Berman,et al.  A standard reference frame for the description of nucleic acid base-pair geometry. , 2001, Journal of molecular biology.

[5]  Yongping Shao,et al.  Biochemical identification of base and phosphate contacts between Fis and a high-affinity DNA binding site. , 2008, Journal of molecular biology.

[6]  R. C. Johnson,et al.  Identification of two functional regions in Fis: the N‐terminus is required to promote Hin‐mediated DNA inversion but not lambda excision. , 1991, The EMBO journal.

[7]  S. McLeod,et al.  The C-terminal domains of the RNA polymerase alpha subunits: contact site with Fis and localization during co-activation with CRP at the Escherichia coli proP P2 promoter. , 2002, Journal of molecular biology.

[8]  S. Neidle Oxford handbook of nucleic acid structure , 1998 .

[9]  Michael A. Crickmore,et al.  Functional Specificity of a Hox Protein Mediated by the Recognition of Minor Groove Structure , 2007, Cell.

[10]  R. Gourse,et al.  Molecular anatomy of a transcription activation patch: FIS–RNA polymerase interactions at the Escherichia coli rrnB P1 promoter , 1997, The EMBO journal.

[11]  D. Perkins-Balding,et al.  Location, degree, and direction of DNA bending associated with the Hin recombinational enhancer sequence and Fis-enhancer complex , 1997, Journal of bacteriology.

[12]  Reid C. Johnson,et al.  Fis targets assembly of the Xis nucleoprotein filament to promote excisive recombination by phage lambda. , 2007, Journal of molecular biology.

[13]  Janez Plavec,et al.  A unified model for the origin of DNA sequence-directed curvature. , 2003, Biopolymers.

[14]  C. Ball,et al.  Dramatic changes in Fis levels upon nutrient upshift in Escherichia coli , 1992, Journal of bacteriology.

[15]  Reid C. Johnson,et al.  The −35 sequence location and the Fis–sigma factor interface determine σS selectivity of the proP (P2) promoter in Escherichia coli , 2007, Molecular microbiology.

[16]  Randy J Read,et al.  Electronic Reprint Biological Crystallography Phenix: Building New Software for Automated Crystallographic Structure Determination Biological Crystallography Phenix: Building New Software for Automated Crystallographic Structure Determination , 2022 .

[17]  A. Travers,et al.  The Escherichia coli FIS protein is not required for the activation of tyrT transcription on entry into exponential growth. , 1993, The EMBO journal.

[18]  J. Feigon,et al.  DNA A-tract bending in three dimensions : Solving the dA 4 T 4 vs . dT 4 A 4 conundrum , 2004 .

[19]  T. D. Schneider,et al.  Information analysis of Fis binding sites. , 1997, Nucleic acids research.

[20]  J. Feigon,et al.  DNA A-tract bending in three dimensions: solving the dA4T4 vs. dT4A4 conundrum. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[21]  W. Colón,et al.  Common and Variable Contributions of Fis Residues to High-Affinity Binding at Different DNA Sequences , 2006, Journal of bacteriology.

[22]  Reid C. Johnson,et al.  Chapter 8:Bending and Compaction of DNA by Proteins , 2008 .

[23]  The molecular structure of wild-type and a mutant Fis protein: relationship between mutational changes and recombinational enhancer function or DNA binding. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[24]  W. Saenger,et al.  Three-dimensional structure of the E. coli DMA-binding protein FIS , 1991, Nature.

[25]  Xiang-Jun Lu,et al.  3DNA: a versatile, integrated software system for the analysis, rebuilding and visualization of three-dimensional nucleic-acid structures , 2008, Nature Protocols.

[26]  R. Hegde The papillomavirus E2 proteins: structure, function, and biology. , 2002, Annual review of biophysics and biomolecular structure.

[27]  R. Mann,et al.  The role of DNA shape in protein-DNA recognition , 2009, Nature.

[28]  R. Kahmann,et al.  The N-terminal part of the E.coli DNA binding protein FIS is essential for stimulating site-specific DNA inversion but is not required for specific DNA binding. , 1991, Nucleic acids research.

[29]  A. Travers,et al.  DNA supercoiling and transcription in Escherichia coli: The FIS connection. , 2001, Biochimie.

[30]  R. C. Johnson,et al.  The Fis protein: it's not just for DNA inversion anymore , 1992, Molecular microbiology.

[31]  T. Steitz,et al.  Correction of X-ray intensities from single crystals containing lattice-translocation defects. , 2005, Acta crystallographica. Section D, Biological crystallography.

[32]  L. Williams,et al.  High-resolution structure of an extended A-tract: [d(CGCAAATTTGCG)]2. , 2004, Journal of the American Chemical Society.

[33]  Helen M Berman,et al.  Signatures of protein-DNA recognition in free DNA binding sites. , 2009, Journal of molecular biology.

[34]  Mark S. Thomas,et al.  Architecture of Fis-activated transcription complexes at the Escherichia coli rrnB P1 and rrnE P1 promoters. , 2002, Journal of molecular biology.

[35]  T. Haran,et al.  The unique structure of A-tracts and intrinsic DNA bending , 2009, Quarterly Reviews of Biophysics.

[36]  Martin Phillips,et al.  Toward the structural genomics of complexes: crystal structure of a PE/PPE protein complex from Mycobacterium tuberculosis. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[37]  G. Murshudov,et al.  Refinement of macromolecular structures by the maximum-likelihood method. , 1997, Acta crystallographica. Section D, Biological crystallography.

[38]  R. C. Johnson,et al.  Localization of amino acids required for Fis to function as a class II transcriptional activator at the RpoS-dependent proP P2 promoter. , 1999, Journal of molecular biology.

[39]  Struther Arnott,et al.  The structure of B-DNA in oriented fibers. , 1996, Journal of biomolecular structure & dynamics.

[40]  H. Yuan,et al.  Structural analysis of the transcriptional activation region on Fis: crystal structures of six Fis mutants with different activation properties. , 2000, Journal of molecular biology.

[41]  H. Rozenberg,et al.  DNA bending by an adenine–thymine tract and its role in gene regulation , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[42]  S Brunak,et al.  Genome organisation and chromatin structure in Escherichia coli. , 2001, Biochimie.

[43]  R. C. Johnson,et al.  Structure of the Escherichia coli Fis-DNA complex probed by protein conjugated with 1,10-phenanthroline copper(I) complex. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[44]  Randy J. Read,et al.  Phaser crystallographic software , 2007, Journal of applied crystallography.

[45]  W. Arber,et al.  Mutational analysis of a prokaryotic recombinational enhancer element with two functions. , 1989, The EMBO journal.

[46]  V. Zhurkin,et al.  DNA sequence-dependent deformability deduced from protein-DNA crystal complexes. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[47]  R. Dickerson,et al.  Intrinsic bending and deformability at the T-A step of CCTTTAAAGG: a comparative analysis of T-A and A-T steps within A-tracts. , 2001, Journal of molecular biology.

[48]  R. Gourse,et al.  The transcriptional activator protein FIS: DNA interactions and cooperative interactions with RNA polymerase at the Escherichia coli rrnB P1 promoter. , 1995, Journal of molecular biology.

[49]  R Lavery,et al.  The definition of generalized helicoidal parameters and of axis curvature for irregular nucleic acids. , 1988, Journal of biomolecular structure & dynamics.

[50]  Yongping Shao,et al.  Functional characterization of the Escherichia coli Fis-DNA binding sequence. , 2008, Journal of molecular biology.

[51]  Byung-Kwan Cho,et al.  Genome-wide analysis of Fis binding in Escherichia coli indicates a causative role for A-/AT-tracts. , 2008, Genome research.

[52]  R J Read,et al.  Crystallography & NMR system: A new software suite for macromolecular structure determination. , 1998, Acta crystallographica. Section D, Biological crystallography.

[53]  Gary Parkinson,et al.  Structural Basis of Transcription Activation: The CAP-αCTD-DNA Complex , 2002, Science.

[54]  R. Dickerson,et al.  Testing water‐mediated DNA recognition by the Hin recombinase , 2002, The EMBO journal.

[55]  W. Olson,et al.  3DNA: a software package for the analysis, rebuilding and visualization of three-dimensional nucleic acid structures. , 2003, Nucleic acids research.

[56]  Kevin Cowtan,et al.  research papers Acta Crystallographica Section D Biological , 2005 .

[57]  C. Dorman Nucleoid-associated proteins and bacterial physiology. , 2009, Advances in applied microbiology.

[58]  Reid C. Johnson,et al.  Major Nucleoid Proteins in the Structure and Function of the Escherichia coli Chromosome , 2005 .

[59]  R. C. Johnson,et al.  Variable structures of Fis-DNA complexes determined by flanking DNA-protein contacts. , 1996, Journal of molecular biology.

[60]  Reid C. Johnson,et al.  The transactivation region of the Fis protein that controls site‐specific DNA inversion contains extended mobile β‐hairpin arms , 1997, The EMBO journal.

[61]  Andrew Travers,et al.  Bacterial chromatin. , 2005, Current opinion in genetics & development.

[62]  Yongli Zhang,et al.  Predicting indirect readout effects in protein-DNA interactions. , 2004, Proceedings of the National Academy of Sciences of the United States of America.