Evaluation of the DNA Binding Tendencies of the Transition State Regulator AbrB

Abstract

Global transition state regulator proteins represent one of the most diverse classes of prokaryotic transcription factors. One such transition state regulator, AbrB from Bacillus subtilis, is known to bind more than 60 gene targets yet displays specificity within this target set by binding each promoter with a different affinity. Microelectrospray ionization mass spectrometry (microESI-MS), circular dichroism, fluorescence, UV spectroscopy, and molecular modeling were used to elucidate differences among AbrB, DNA, and AbrB-DNA complexes. MicroESI-MS analysis of AbrB confirmed its stable macromolecular state as being tetrameric and verified the same stoichiometric state in complex with DNA targets. MicroESI-MS, circular dichroism, and fluorescence provided relative binding affinities for AbrB-DNA interactions in a qualitative manner. UV spectroscopy was used in a quantitative manner to determine solution phase dissociation constants for AbrB-DNA complexes. General DNA structural parameters for all known natural AbrB binding sequences were also studied and significant similarities in topological constraints (stretch, opening, and propeller twist) were observed. It is likely that these parameters contribute to the differential binding proclivities of AbrB. In addition to providing an improved understanding of transition state regulator-DNA binding properties and structural tendencies of target promoters, this comprehensive and corroborative spectroscopic study endorses the use of microESI-MS for rapidly ascertaining qualitative binding trends in noncovalent systems in a high-throughput manner.

Transition State Regulator AbrB Inhibits Transcription of Bacillus amyloliquefaciens FZB45 Phytase through Binding at Two Distinct Sites Located within the Extended phyC Promoter Region

“…Local variations of the DNA helix configurations (e.g., minor groove width and degree of propeller twisting, etc.) contribute to the differential binding proclivities of AbrB (3). Here, we demonstrated that the replacement of local AT-rich sequence stretches within the AbrB binding region by GC-rich oligonucleotides decreases affinity toward AbrB.…”

“…The functional form of AbrB has been described as a homotetramer rather than as a homodimer (2,20). DNAbinding and dimerization functions are located in the N-terminal domain (AbrBN), and the C-terminal domain is responsible for the tendency towards multimerization (2,3,20,21). In this study, we have not addressed the oligomeric state of AbrB, but gel filtration suggested that AbrB could form oligomers containing more than four subunits (see Fig.…”

An AbrB-Like Protein Regulates the Expression of the Bidirectional Hydrogenase in Synechocystis sp. Strain PCC 6803

“…During in silico analyzation of the DNA sequence of the two promoter regions, it was not possible to find putative recognition motifs, since a search for highly similar DNA stretches failed to give possible hits. Interestingly, AbrB from B. subtilis is well known for its significant promiscuity in DNA recognition (6,7,42) and the comparison between the described target regions reveals no apparent base sequence that can be defined as an AbrB consensus binding site (48).…”

Abh and AbrB Control of Bacillus subtilis Antimicrobial Gene Expression

“…High-affinity sites of AbrB-DNA interactions selected using in vitro methods do show convergence to a consensus, but sequences resembling this consensus are rarely found in the chromosomal sites (50, 53). It has been hypothesized that AbrB achieves binding specificity by recognizing three-dimensional DNA architectures that are shared by a finite subset of base sequences (5,40,42,45,53). A major factor accounting for what has been termed the "limited promiscuity" of AbrB-DNA interactions is believed to be the dynamic flexibility in the DNA-binding domain of the protein; this flexibility allows it to conform to different targets with thermodynamically favorable contacts (4, 6, 47).…”