The Aspergillus nidulans transcription factor AlcR forms a stable complex with its half‐site DNA: a NMR study

Abstract

The Aspergillus nidulans transcription factor AlcR is shown by NMR and gel retardation assay to form a stable complex with oligonucleotide sequences comprising the consensus half-site 5'-TGCGG-3'. Apparent /jM dissociation constants are evaluated by both methods. The measured lifetime of the complex is 74 ±7 ms at 20°C with the following DNA sequence: 5'-C1G2T3G4C5G6G7A8T9C10-3'. The major chemical shift variations upon binding involve both the two adjacent GC pairs (G6 and G7) and, clearly, the AT pairs at both ends of the consensus sequence (T3 and A8), suggesting additional contacts of the protein with the DNA. This extensive and strong interaction with the half-site is another example of the variability in contacts of the fungal DNA-binding proteins containing Zn 2 Cys 6 domains with their consensus sites. It is the first demonstration that a binuclear cluster protein can bind to DNA as a monomer with strong affinity.

The Regulator of Nitrate Assimilation in Ascomycetes Is a Dimer Which Binds a Nonrepeated, Asymmetrical Sequence

“…Other work from this laboratory has already shown that the zinc binuclear cluster AlcR protein can bind as a monomer (8,22) and that the single sites play physiological roles in vivo (27). Residues of a monomer located in different places along the DNA binding domain could recognize the two half-sites of the NirA binding sequence.…”

“…Furthermore, AlcR has been reported to recognize direct, inverted, and everted repeats, with the last two separated by a variable number of base pairs. The solution to this apparent paradox is simple: AlcR is able to bind as a monomer (8,18,19,22).…”

Unique DNA Binding Specificity of the Binuclear Zinc AlcR Activator of the Ethanol Utilization Pathway in Aspergillus nidulans

“…However, AlcR is distinguished by a number of major features from the other proteins of this class as will be discussed later. These features most probably account for the unique AlcR specificity which is characterized by in vitro binding to single sites (5,6,9), containing the consensus core 5Ј-(T/A)GCGG-3Ј and, in vivo by the functional relevance of these sites, organized in repeated elements, which are defined as UASalc (3,4,6,27).…”

“…Transcriptional regulation is mediated by the binding of AlcR to different cognate sites, encompassing the same consensus core, 5Ј-(T/A)GCGG-3Ј, organized as direct and inverted repeats, located upstream of these genes (3)(4)(5)(6). AlcR is a fungal transcription factor of the zinc binuclear cluster family, containing six cysteines and two zinc atoms (C6 protein class) (7)(8)(9). However, AlcR is distinguishable from other proteins in this family by its structural and binding properties.…”

“…NMR studies have shown that the isolated AlcR zinc binuclear cluster domain (residues 1-60) binds to a single copy site as a monomer, albeit with a low affinity (9). More recently, binding experiments performed with a longer AlcR protein , containing the region downstream of the zinc cluster, have clearly indicated that one AlcR molecule binds with high affinity to DNA single sites (5 ϫ 10 Ϫ8 M) (5,6).…”