Linker length and composition influence the flexibility of Oct-1 DNA binding
Open Access
- 15 April 1997
- journal article
- research article
- Published by Springer Nature in The EMBO Journal
- Vol. 16 (8), 2043-2053
- https://doi.org/10.1093/emboj/16.8.2043
Abstract
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.Keywords
This publication has 37 references indexed in Scilit:
- Oct-1 POU domain-DNA interactions: cooperative binding of isolated subdomains and effects of covalent linkage.Genes & Development, 1996
- Crystal Structure of the MATa1/MATα2 Homeodomain Heterodimer Bound to DNAScience, 1995
- Site-specific conformational alteration of the Oct-1 POU domain-DNA complex as the basis for differential recognition by Vmw65 (VP16)Cell, 1994
- An Invariant Asparagine in the POU-Specific Homeodomain Regulates the Specificity of the Oct-2 POU MotifBiochemistry, 1994
- Short Introns Interrupting the Oct-2 POU Domain May Prevent Recombination between POU Family Genes without Interfering with Potential POU Domain ‘Shuffling’ in EvolutionBiological Chemistry Hoppe-Seyler, 1994
- Solution structure of the POU-specific DNA-binding domain of Oct-1Nature, 1993
- A single amino acid exchange transfers VP16-induced positive control from the Oct-1 to the Oct-2 homeo domain.Genes & Development, 1992
- Biochemical characterization of the Oct-2 POU domain with implications for bipartite DNA recognitionBiochemistry, 1992
- Octamania: The POU factors in murine developmentTrends in Genetics, 1991
- A tissue-specific transcription factor containing a homeodomain specifies a pituitary phenotypeCell, 1988