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Structural analysis of the DNA target site and its interaction with Mbp1

Structural analysis of the DNA target site and its interaction with Mbp1
Structural analysis of the DNA target site and its interaction with Mbp1
The solution structure of a 14 base-pair non-self complementary DNA duplex containing the consensus-binding site of the yeast transcription factor Mbp1 has been determined by NMR using a combination of scalar coupling analysis, time-dependent NOEs, residual dipolar couplings and 13C-edited NMR spectroscopy of a duplex prepared with one strand uniformly labeled with 13C-nucleotides. As expected, the free DNA duplex is within the B-family of structures, and within experimental limits is straight. However, there are clear local structural variations associated with the consensus CGCG element in the binding sequence that are important for sequence recognition. In the complex, the DNA bends around the protein, which also undergoes some conformational rearrangement in the C-terminal region. Structural constraints derived from paramagnetic perturbation experiments with spin-labeled DNA, chemical shift perturbation experiments of the DNA, previous cross-saturation, chemical shift perturbation experiments on the protein, information from mutational analysis, and electrostatics calculations have been used to produce a detailed docked structure using the known solution conformation of the free protein and other spectroscopic information about the Mbp1:DNA complex. A Monte Carlo-based docking procedure with restrained MD in a fully solvated system subjected to available experimental constraints produced models that account for the available structural data, and can rationalize the extensive thermodynamic data about the Mbp1:DNA complex. The protein:DNA interface is closely packed and is associated with a small number of specific contacts. The structure shows an extensive positively charged surface that accounts for the high polyelectrolyte contribution to binding.
1477-0520
4981-4991
Chernatynskaya, Anna V.
f73cfa0c-b94f-4a5a-919f-e23bd239c57a
Deleeuw, Lynn
d6c33107-6a7c-4f80-a799-b3b784c35c1d
Trent, John O.
aabd4141-64bf-4107-9e61-e1407c09d430
Brown, Tom
a64aae36-bb30-42df-88a2-11be394e8c89
Lane, Andrew N.
45ad386f-2308-4610-998b-7913fa4d457c
Chernatynskaya, Anna V.
f73cfa0c-b94f-4a5a-919f-e23bd239c57a
Deleeuw, Lynn
d6c33107-6a7c-4f80-a799-b3b784c35c1d
Trent, John O.
aabd4141-64bf-4107-9e61-e1407c09d430
Brown, Tom
a64aae36-bb30-42df-88a2-11be394e8c89
Lane, Andrew N.
45ad386f-2308-4610-998b-7913fa4d457c

Chernatynskaya, Anna V., Deleeuw, Lynn, Trent, John O., Brown, Tom and Lane, Andrew N. (2009) Structural analysis of the DNA target site and its interaction with Mbp1. Organic & Biomolecular Chemistry, 7 (23), 4981-4991. (doi:10.1039/b912309a).

Record type: Article

Abstract

The solution structure of a 14 base-pair non-self complementary DNA duplex containing the consensus-binding site of the yeast transcription factor Mbp1 has been determined by NMR using a combination of scalar coupling analysis, time-dependent NOEs, residual dipolar couplings and 13C-edited NMR spectroscopy of a duplex prepared with one strand uniformly labeled with 13C-nucleotides. As expected, the free DNA duplex is within the B-family of structures, and within experimental limits is straight. However, there are clear local structural variations associated with the consensus CGCG element in the binding sequence that are important for sequence recognition. In the complex, the DNA bends around the protein, which also undergoes some conformational rearrangement in the C-terminal region. Structural constraints derived from paramagnetic perturbation experiments with spin-labeled DNA, chemical shift perturbation experiments of the DNA, previous cross-saturation, chemical shift perturbation experiments on the protein, information from mutational analysis, and electrostatics calculations have been used to produce a detailed docked structure using the known solution conformation of the free protein and other spectroscopic information about the Mbp1:DNA complex. A Monte Carlo-based docking procedure with restrained MD in a fully solvated system subjected to available experimental constraints produced models that account for the available structural data, and can rationalize the extensive thermodynamic data about the Mbp1:DNA complex. The protein:DNA interface is closely packed and is associated with a small number of specific contacts. The structure shows an extensive positively charged surface that accounts for the high polyelectrolyte contribution to binding.

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Published date: December 2009

Identifiers

Local EPrints ID: 146663
URI: https://eprints.soton.ac.uk/id/eprint/146663
ISSN: 1477-0520
PURE UUID: e5ca8ecf-68be-45ec-8f89-b8c5d90ff9af

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Date deposited: 22 Apr 2010 08:38
Last modified: 17 Jul 2019 00:04

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