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DNA sequence-specific recognition by a transcriptional regulator requires indirect readout of A-tracts.

ABSTRACT: The bacteriophage Ø29 transcriptional regulator p4 binds to promoters of different intrinsic activities. The p4-DNA complex contains two identical protomers that make similar interactions with the target sequence 5'-AACTTTTT-15 bp-AAAATGTT-3'. To define how the various elements in the target sequence contribute to p4's affinity, we studied p4 binding to a series of mutated binding sites. The binding specificity depends critically on base pairs of the target sequence through both direct as well as indirect readout. There is only one specific contact between a base and an amino acid residue; other contacts take place with the phosphate backbone. Alteration of direct amino acid-base contacts, or mutation of non-contacted A.T base pairs at A-tracts abolished binding. We generated three 5 ns molecular dynamics (MD) simulations to investigate the basis for the p4-DNA complex specificity. Recognition is controlled by the protein and depends on DNA dynamic properties. MD results on protein-DNA contacts and the divergence of p4 affinity to modified binding sites reveal an inherent asymmetry, which is required for p4-specific binding and may be crucial for transcription regulation.

SUBMITTER: Mendieta J

PROVIDER: S-EPMC1904284 | biostudies-literature | 2007

REPOSITORIES: biostudies-literature

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DNA sequence-specific recognition by a transcriptional regulator requires indirect readout of A-tracts.

Mendieta Jesús J Mendieta Jesús J Pérez-Lago Laura L Salas Margarita M Camacho Ana A

Nucleic acids research 20070422 10

The bacteriophage Ø29 transcriptional regulator p4 binds to promoters of different intrinsic activities. The p4-DNA complex contains two identical protomers that make similar interactions with the target sequence 5'-AACTTTTT-15 bp-AAAATGTT-3'. To define how the various elements in the target sequence contribute to p4's affinity, we studied p4 binding to a series of mutated binding sites. The binding specificity depends critically on base pairs of the target sequence through both direct as well a ...[more]

PMID: 17452358

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Project description:The catabolite activator protein (CAP) bends DNA in the CAP-DNA complex, typically introducing a sharp DNA kink, with a roll angle of approximately 40 degrees and a twist angle of approximately 20 degrees, between positions 6 and 7 of the DNA half-site, 5'-A1A2A3T4G5T6G7A8T9C10T11 -3' ("primary kink"). In previous work, we showed that CAP recognizes the nucleotide immediately 5' to the primary-kink site, T6, through an "indirect-readout" mechanism involving sequence effects on energetics of primary-kink formation. Here, to understand further this example of indirect readout, we have determined crystal structures of CAP-DNA complexes containing each possible nucleotide at position 6. The structures show that CAP can introduce a DNA kink at the primary-kink site with any nucleotide at position 6. The DNA kink is sharp with the consensus pyrimidine-purine step T6G7 and the non-consensus pyrimidine-purine step C6G7 (roll angles of approximately 42 degrees, twist angles of approximately 16 degrees ), but is much less sharp with the non-consensus purine-purine steps A6G7 and G6G7 (roll angles of approximately 20 degrees, twist angles of approximately 17 degrees). We infer that CAP discriminates between consensus and non-consensus pyrimidine-purine steps at positions 6-7 solely based on differences in the energetics of DNA deformation, but that CAP discriminates between the consensus pyrimidine-purine step and non-consensus purine-purine steps at positions 6-7 both based on differences in the energetics of DNA deformation and based on qualitative differences in DNA deformation. The structures further show that CAP can achieve a similar, approximately 46 degrees per DNA half-site, overall DNA bend through a sharp DNA kink, a less sharp DNA kink, or a smooth DNA bend. Analysis of these and other crystal structures of CAP-DNA complexes indicates that there is a large, approximately 28 degrees per DNA half-site, out-of-plane component of CAP-induced DNA bending in structures not constrained by end-to-end DNA lattice interactions and that lattice contacts involving CAP tend to involve residues in or near biologically functional surfaces.

Dataset Information

DNA sequence-specific recognition by a transcriptional regulator requires indirect readout of A-tracts.

Publications

DNA sequence-specific recognition by a transcriptional regulator requires indirect readout of A-tracts.

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