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EMBO J. Apr 15, 1997; 16(8): 2043–2053.
PMCID: PMC1169807

Linker length and composition influence the flexibility of Oct-1 DNA binding.


POU domain transcription factors have two separate helix-turn-helix DNA-binding subdomains, the POU homeodomain (POUhd) and the POU-specific domain (POUs). Each subdomain recognizes a specific subsite of 4 or 5 bp in the octamer recognition sequence. The Oct-1 POU subdomains are connected by a 23 amino acid unstructured linker region. To investigate the requirements for the linker and its role in DNA recognition, we constructed POU domains in which the subdomains are connected with linkers varying in length between 2 and 37 amino acids. Binding to the natural octamer site required a minimal linker length of between 10 and 14 amino acids. A POU domain with an eight amino acid linker, however, had a high affinity for a site in which the POUs recognition sequence was inverted. Computer modelling shows that inversion of the POUs subdomain shortens the distance between the subdomains sufficiently to enable an eight amino acid linker to bridge the distance. DNase I footprinting as well as mutation of the POUs-binding site confirms the inverted orientation of the POUs domain. Switching of the POUs and POUhd subdomains and separation by 3 bp leads to a large distance which could only be bridged effectively by a long 37 amino acid linker. In addition to linker length, mutation of a conserved glutamate residue in the linker affected binding. As shown by surface plasmon resonance measurements, this was caused by a decrease in the on-rate. Our data indicate that there are both length and sequence requirements in the linker region which allow flexibility leading to selective binding to differently spaced and oriented subsites.

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Selected References

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  • Botfield MC, Jancso A, Weiss MA. An invariant asparagine in the POU-specific homeodomain regulates the specificity of the Oct-2 POU motif. Biochemistry. 1994 Jul 5;33(26):8113–8121. [PubMed]
  • Certel K, Anderson MG, Shrigley RJ, Johnson WA. Distinct variant DNA-binding sites determine cell-specific autoregulated expression of the Drosophila POU domain transcription factor drifter in midline glia or trachea. Mol Cell Biol. 1996 Apr;16(4):1813–1823. [PMC free article] [PubMed]
  • Cleary MA, Herr W. Mechanisms for flexibility in DNA sequence recognition and VP16-induced complex formation by the Oct-1 POU domain. Mol Cell Biol. 1995 Apr;15(4):2090–2100. [PMC free article] [PubMed]
  • Dekker N, Cox M, Boelens R, Verrijzer CP, van der Vliet PC, Kaptein R. Solution structure of the POU-specific DNA-binding domain of Oct-1. Nature. 1993 Apr 29;362(6423):852–855. [PubMed]
  • Greenstein D, Hird S, Plasterk RH, Andachi Y, Kohara Y, Wang B, Finney M, Ruvkun G. Targeted mutations in the Caenorhabditis elegans POU homeo box gene ceh-18 cause defects in oocyte cell cycle arrest, gonad migration, and epidermal differentiation. Genes Dev. 1994 Aug 15;8(16):1935–1948. [PubMed]
  • Gstaiger M, Knoepfel L, Georgiev O, Schaffner W, Hovens CM. A B-cell coactivator of octamer-binding transcription factors. Nature. 1995 Jan 26;373(6512):360–362. [PubMed]
  • Gstaiger M, Georgiev O, van Leeuwen H, van der Vliet P, Schaffner W. The B cell coactivator Bob1 shows DNA sequence-dependent complex formation with Oct-1/Oct-2 factors, leading to differential promoter activation. EMBO J. 1996 Jun 3;15(11):2781–2790. [PMC free article] [PubMed]
  • Herr W, Cleary MA. The POU domain: versatility in transcriptional regulation by a flexible two-in-one DNA-binding domain. Genes Dev. 1995 Jul 15;9(14):1679–1693. [PubMed]
  • Ingraham HA, Chen RP, Mangalam HJ, Elsholtz HP, Flynn SE, Lin CR, Simmons DM, Swanson L, Rosenfeld MG. A tissue-specific transcription factor containing a homeodomain specifies a pituitary phenotype. Cell. 1988 Nov 4;55(3):519–529. [PubMed]
  • Jin Y, Mead J, Li T, Wolberger C, Vershon AK. Altered DNA recognition and bending by insertions in the alpha 2 tail of the yeast a1/alpha 2 homeodomain heterodimer. Science. 1995 Oct 13;270(5234):290–293. [PubMed]
  • Klemm JD, Pabo CO. Oct-1 POU domain-DNA interactions: cooperative binding of isolated subdomains and effects of covalent linkage. Genes Dev. 1996 Jan 1;10(1):27–36. [PubMed]
  • Klemm JD, Rould MA, Aurora R, Herr W, Pabo CO. Crystal structure of the Oct-1 POU domain bound to an octamer site: DNA recognition with tethered DNA-binding modules. Cell. 1994 Apr 8;77(1):21–32. [PubMed]
  • Lai JS, Cleary MA, Herr W. A single amino acid exchange transfers VP16-induced positive control from the Oct-1 to the Oct-2 homeo domain. Genes Dev. 1992 Nov;6(11):2058–2065. [PubMed]
  • Li P, He X, Gerrero MR, Mok M, Aggarwal A, Rosenfeld MG. Spacing and orientation of bipartite DNA-binding motifs as potential functional determinants for POU domain factors. Genes Dev. 1993 Dec;7(12B):2483–2496. [PubMed]
  • Li T, Stark MR, Johnson AD, Wolberger C. Crystal structure of the MATa1/MAT alpha 2 homeodomain heterodimer bound to DNA. Science. 1995 Oct 13;270(5234):262–269. [PubMed]
  • Liang J, Moye-Rowley S, Maurer RA. In vivo mutational analysis of the DNA binding domain of the tissue-specific transcription factor, Pit-1. J Biol Chem. 1995 Oct 27;270(43):25520–25525. [PubMed]
  • Andrés V, Chiara MD, Mahdavi V. A new bipartite DNA-binding domain: cooperative interaction between the cut repeat and homeo domain of the cut homeo proteins. Genes Dev. 1994 Jan;8(2):245–257. [PubMed]
  • Matsuo K, Clay O, Künzler P, Georgiev O, Urbánek P, Schaffner W. Short introns interrupting the Oct-2 POU domain may prevent recombination between POU family genes without interfering with potential POU domain 'shuffling' in evolution. Biol Chem Hoppe Seyler. 1994 Oct;375(10):675–683. [PubMed]
  • Aurora R, Herr W. Segments of the POU domain influence one another's DNA-binding specificity. Mol Cell Biol. 1992 Feb;12(2):455–467. [PMC free article] [PubMed]
  • Ogata K, Morikawa S, Nakamura H, Sekikawa A, Inoue T, Kanai H, Sarai A, Ishii S, Nishimura Y. Solution structure of a specific DNA complex of the Myb DNA-binding domain with cooperative recognition helices. Cell. 1994 Nov 18;79(4):639–648. [PubMed]
  • O'Shannessy DJ, Brigham-Burke M, Soneson KK, Hensley P, Brooks I. Determination of rate and equilibrium binding constants for macromolecular interactions using surface plasmon resonance: use of nonlinear least squares analysis methods. Anal Biochem. 1993 Aug 1;212(2):457–468. [PubMed]
  • Pavletich NP, Pabo CO. Crystal structure of a five-finger GLI-DNA complex: new perspectives on zinc fingers. Science. 1993 Sep 24;261(5129):1701–1707. [PubMed]
  • Pomerantz JL, Kristie TM, Sharp PA. Recognition of the surface of a homeo domain protein. Genes Dev. 1992 Nov;6(11):2047–2057. [PubMed]
  • Pomerantz JL, Sharp PA, Pabo CO. Structure-based design of transcription factors. Science. 1995 Jan 6;267(5194):93–96. [PubMed]
  • Schöler HR. Octamania: the POU factors in murine development. Trends Genet. 1991 Oct;7(10):323–329. [PubMed]
  • Strubin M, Newell JW, Matthias P. OBF-1, a novel B cell-specific coactivator that stimulates immunoglobulin promoter activity through association with octamer-binding proteins. Cell. 1995 Feb 10;80(3):497–506. [PubMed]
  • Studier FW, Rosenberg AH, Dunn JJ, Dubendorff JW. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 1990;185:60–89. [PubMed]
  • Sturm RA, Cassady JL, Das G, Romo A, Evans GA. Chromosomal structure and expression of the human OTF1 locus encoding the Oct-1 protein. Genomics. 1993 May;16(2):333–341. [PubMed]
  • van Leeuwen HC, Strating MJ, Cox M, Kaptein R, van der Vliet PC. Mutation of the Oct-1 POU-specific recognition helix leads to altered DNA binding and influences enhancement of adenovirus DNA replication. Nucleic Acids Res. 1995 Aug 25;23(16):3189–3197. [PMC free article] [PubMed]
  • Verrijzer CP, Kal AJ, van der Vliet PC. The oct-1 homeo domain contacts only part of the octamer sequence and full oct-1 DNA-binding activity requires the POU-specific domain. Genes Dev. 1990 Nov;4(11):1964–1974. [PubMed]
  • Verrijzer CP, Alkema MJ, van Weperen WW, Van Leeuwen HC, Strating MJ, van der Vliet PC. The DNA binding specificity of the bipartite POU domain and its subdomains. EMBO J. 1992 Dec;11(13):4993–5003. [PMC free article] [PubMed]
  • Verrijzer CP, van Oosterhout JA, van Weperen WW, van der Vliet PC. POU proteins bend DNA via the POU-specific domain. EMBO J. 1991 Oct;10(10):3007–3014. [PMC free article] [PubMed]
  • Verrijzer CP, Strating M, Mul YM, van der Vliet PC. POU domain transcription factors from different subclasses stimulate adenovirus DNA replication. Nucleic Acids Res. 1992 Dec 11;20(23):6369–6375. [PMC free article] [PubMed]
  • Walker S, Hayes S, O'Hare P. Site-specific conformational alteration of the Oct-1 POU domain-DNA complex as the basis for differential recognition by Vmw65 (VP16). Cell. 1994 Dec 2;79(5):841–852. [PubMed]

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