Abstract
AlcR is the transcriptional activator in Aspergillus nidulans, necessary for the induction of the alc gene cluster. It belongs to the Zn2Cys6 zinc cluster protein family, but contains some striking differences compared with other proteins of this group. In this report, we show that no dimerization element is present in the entire AlcR protein which occurs in solution as a monomer and binds also to its cognate sites as a monomer. Another important feature of AlcR is its unique specificity for single sites occurring naturally as inverted or direct repeats and sharing a common motif, 5'-(T/A)GCGG-3'. Like most other Zn2Cys6 proteins, AlcR contacts directly with the CGG triplet and, in addition, the upstream adjacent guanine is required for high affinity binding. We also establish that the flanking regions outside the core play an essential role in tight binding. From our in vitro analysis, we propose an optimal AlcR-binding site which is 5'-PuNGCGG-AT rich 3'.
Highlights
The Aspergillus nidulans protein, AlcR, activates transcription of genes required for the oxidation of ethanol and other alc clustered genes whose functions are currently being studied [1, 2]
AlcR is distinguishable from other proteins in this family by its structural and binding properties. It has an asymmetric structure resulting from an additional 16 residues between the third and the fourth cysteines compared with 6 – 8 residues usually found in the other proteins of this class [7]. In this loop, the proline residue conserved in all the zinc binuclear cluster domains and which was shown to be important for zinc binding, as stated for GAL4 [10] and more recently for PrnA [11], is not present in AlcR
These differences have no effect on the distance between the two zinc atoms and between zinc and sulfydryl ligands of cysteine [12] which are similar to those found in the solved structure of GAL4, PPR1, and PUT3 [13,14,15,16]
Summary
Plasmids and Phages—Escherichia coli strains CSH50: ⌬(proLac) FϪ(proABLacIqZ) ⌬M15, traD36 was used in phage immunity tests. DNA Binding Specificity of AlcR Binuclear Cluster amplified by polymerase chain reaction primed from specific oligonucleotides containing SalI sites at both ends and cloned into the pC132 plasmid cut by SalI. In all these constructs the AlcR domains were fused in-frame with the phage CI repressor NH2-terminal domain at the 5Ј end and the -galactosidase ␣-peptide gene at the 3Ј end. Electrophoretic Mobility Shift Assays—Oligonucleotide probes containing either direct repeat targets a or c or inverted repeat target b from the alcA promoter or an artificial single copy site sc and its mutated derivatives were used in most gel shift experiments. Muro-Pastor. 3–20 l of translation products were incubated for 1 h with 0.005% glutaraldehyde in 50 mM potassium phosphate buffer, pH 7.5, and afterward analyzed either by 8 or 10% SDS-polyacrylamide gel electrophoresis followed by fluorography
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