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DNA-Binding Properties of AbrB: Relating Structure to Function

M.A. Strauch

The DNA-binding protein AbrB is a global transcriptional regulator of numerous Bacillus subtilis genes that are expressed in response to nutrient limitation and other environmental stresses. More than 40 sites on the bacterial chromosome with specific AbrB-binding affinity have been detected, but a variety of evidence suggests many more probably exist. The sequence heterogeneity of these regions, coupled with results of examinations of optimal binding sites selected in vitro, indicates that AbrB has some form of inherent structural flexibility that allows it to bind with high affinity to these disparate sequences. The global control exerted by AbrB is a consequence of its ability to achieve regulatory effects within a variety of sequence contexts.

In solution, AbrB appears to be a tetramer of identical 10.5-kD subunits that assumes some type of nonglobular shape. DNA-binding determinants are confined to the N-terminal domains of the constituent monomers, but 2 of these identical domains in a proper spatial configuration are necessary for binding specificity and high affinity. The C-terminal domains are primarily responsible for the multimerization reaction that positions N-terminal domains in correct proximity for DNA binding.

To investigate the biophysical basis of the DNA-binding properties of AbrB, in collaboration with J. Cavanagh, New York State Department of Health, we are using nuclear magnetic resonance techniques to determine the structure of the N-terminal domain. Backbone resonance assignments were used to calculate an initial fold for the domain, which seemingly has no resemblance to other previously elucidated DNA-binding motifs. The single -helical region in the N-terminal domain is probably important in contacting DNA, because mutations in this helix completely destroy DNA binding but have no effect on multimeric structure. The nuclear magnetic resonance data also indicate that AbrB has conformation flexibility on either side of the helix, which could lead to alternative spatial positionings of the helix and thus be an important factor in the ability of the protein to interact with the observed heterogeneity of its DNA targets.

We are beginning studies to determine the structure of dimerized N-terminal domains, both free in solution and complexed to suitable DNA target sequences. Site-directed changes in amino acid residues important for structural flexibility and necessary for contact with DNA are being constructed and tested for biochemical properties and in vivo phenotypes. Additionally, mutational and chemical studies on the properties of the C-terminal domain are being done, and we are continuing a variety of investigations designed to elucidate the mechanistic influences of AbrB-DNA complexes on transcriptional efficiency.