Interaction of the trp repressor with trp operator DNA fragments

Beckmann, Pamela; Martin, Stephen R.; Lane, Andrew N.

doi:10.1007/bf00185869

Cited by 8 publications

(2 citation statements)

References 27 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Thermodynamics and structural studies on the ATF-2 and 16mer/ATF-2 complex are currently in progress (M.R.Conte, G.Bloomberg and A.N.Lane, unpublished results), in order to investigate in detail the nature of the recognition process and to define the DNA conformation in the complex. DNA bends are often manifest in the 31 P NMR spectrum (57,58) or of the exchangeable protons (6). Preliminary 31 P and 1 H NMR results on the 16mer/ATF-2 complex (data not shown, M.R.Conte, G.Bloomberg and A.N.Lane, data to be published elsewhere) suggests that the DNA is not greatly distorted in the complex with ATF-2.…”

Section: Discussionmentioning

confidence: 91%

Solution Structure of the ATF-2 Recognition Site and Its Interaction with the ATF-2 Peptide

Conte¹,

Lane²,

Bloomberg

1997

Nucleic Acids Research

View full text Add to dashboard Cite

The effect of leucine zipper proteins binding to the DNA recognition site is controversial. Results from crystallography, gel and solution methods have led to opposite conclusions about the conformation of the DNA in the complex. The role of the DNA binding site in the recognition process and in the gene induction mediated by transcription factors needs to be investigated further. In this article the self-complementary 16 bp oligodeoxynucleotide (CATGTGACGTCACATG)2, which contains the cAMP response element recognised by numerous transcription factors of the leucine zipper family, has been examined free from proteins and in its interaction with the mammalian activating transcription factor 2. The recognition process has been investigated by circular dichroism analysis, which has revealed conformational changes in both DNA and protein upon binding. The solution structure of the 16mer, important in order to define the effects induced by binding of leucine zipper proteins and the intrisic bending properties of DNA, has been determined from NMR data using direct refinement against NOE intensities, analysis of scalar coupling constants and restrained molecular dynamics calculations. Final structures starting from the A and B forms of DNA agreed to a pairwise root mean square deviation (r.m.s.d.) of 1.04 +/- 0.3 A (0.7 +/- 0.2 A to the average) for all atoms. The terminal base pairs were less well determined, and the pairwise deviation of the 12 core bp was 0.83 +/- 0.27 A (0.55 +/- 0.19 A to the average). The final structures are within the B-family with an average helical twist of 36+/-2 degrees. No significant intrinsic DNA bend is shown in the activating transcription factor regulatory site. However, there are substantial deviations from the canonical B-DNA (r.m.s.d. = 3.6 A) in the core of the molecule, associated with relatively large base inclinations.

show abstract

Section: Discussionmentioning

confidence: 91%

Solution Structure of the ATF-2 Recognition Site and Its Interaction with the ATF-2 Peptide

Conte¹,

Lane²,

Bloomberg

1997

Nucleic Acids Research

View full text Add to dashboard Cite

show abstract

“…In addition, there are four peaks upfield and two peaks downfield of the main group. The appearance of both upfield-and downfield-shifted 31 P NMR peaks in DNA-protein and DNA-ligand complexes has been observed in a number of systems (35)(36)(37). It has been suggested that these shifts are associated with changes in backbone torsion angles such as the B I to B II conformation transition (38) and other conformations (39,40).…”

Section: Determination Of the Minimal Dna-binding Domains Ofmentioning

confidence: 99%

Characterization of the DNA-Binding Domains from the Yeast Cell-Cycle Transcription Factors Mbp1 and Swi4

Taylor¹,

McIntosh²,

Pala³

et al. 2000

Biochemistry

View full text Add to dashboard Cite

The minimal DNA-binding domains of the Saccharomyces cerevisiae transcription factors Mbp1 and Swi4 have been identified and their DNA binding properties have been investigated by a combination of methods. An approximately 100 residue region of sequence homology at the N-termini of Mbp1 and Swi4 is necessary but not sufficient for full DNA binding activity. Unexpectedly, nonconserved residues C-terminal to the core domain are essential for DNA binding. Proteolysis of Mbp1 and Swi4 DNA-protein complexes has revealed the extent of these sequences, and C-terminally extended molecules with substantially enhanced DNA binding activity compared to the core domains alone have been produced. The extended Mbp1 and Swi4 proteins bind to their cognate sites with similar affinity [K(A) approximately (1-4) x 10(6) M(-)(1)] and with a 1:1 stoichiometry. However, alanine substitution of two lysine residues (116 and 122) within the C-terminal extension (tail) of Mbp1 considerably reduces the apparent affinity for an MCB (MluI cell-cycle box) containing oligonucleotide. Both Mbp1 and Swi4 are specific for their cognate sites with respect to nonspecific DNA but exhibit similar affinities for the SCB (Swi4/Swi6 cell-cycle box) and MCB consensus elements. Circular dichroism and (1)H NMR spectroscopy reveal that complex formation results in substantial perturbations of base stacking interactions upon DNA binding. These are localized to a central 5'-d(C-A/G-CG)-3' region common to both MCB and SCB sequences consistent with the observed pattern of specificity. Changes in the backbone amide proton and nitrogen chemical shifts upon DNA binding have enabled us to experimentally define a DNA-binding surface on the core N-terminal domain of Mbp1 that is associated with a putative winged helix-turn-helix motif. Furthermore, significant chemical shift differences occur within the C-terminal tail of Mbp1, supporting the notion of two structurally distinct DNA-binding regions within these proteins.

show abstract

Mutants in position 69 of the Trp repressor ofEscherichia coli K12 with altered DNA-binding specificity

Güneş

Müller‐Hill

1996

Molec. Gen. Genet.

View full text Add to dashboard Cite

Structural analysis by X-ray crystallography has indicated that direct contact occurs between Arg69, the second residue of the first helix of the helix-turnhelix (HTH) motif of the Trp repressor, and guanine in position 9 of the alpha-centred consensus trp operator. We therefore replaced residue 69 of the Trp repressor with Gly, Ile, Leu or Gln and tested the resultant repressor mutants for their binding to synthetic symmetrical alpha- or beta-centred trp operator variants, in vivo and in vitro. We present genetic and biochemical evidence that Ile in position 69 of the Trp repressor interacts specifically with thymine in position 9 of the alpha-centred trp operator. There are also interactions with other bases in positions 8 and 9 of the alpha-centred trp operator. In vitro, the Trp repressor of mutant RI69 binds to the consensus alpha-centred trp operator and a similar trp operator variant that carries a T in position 9. In vivo analysis of the interactions of Trp repressor mutant RI69 with symmetrical variants of the beta-centred trp operator shows a change in the specificity of binding to a beta-centred symmetrical trp operator variant with a gua-nine to thymine substitution in position 5, which corresponds to position 9 of the alpha-centred trp operator.

show abstract

Interaction of the trp repressor with trp operator DNA fragments

Cited by 8 publications

References 27 publications

Solution Structure of the ATF-2 Recognition Site and Its Interaction with the ATF-2 Peptide

Solution Structure of the ATF-2 Recognition Site and Its Interaction with the ATF-2 Peptide

Characterization of the DNA-Binding Domains from the Yeast Cell-Cycle Transcription Factors Mbp1 and Swi4

Mutants in position 69 of the Trp repressor ofEscherichia coli K12 with altered DNA-binding specificity

Contact Info

Product

Resources

About