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The mechanism by which the naturally occurring ligand for a nuclear hormone receptor regulates transcription remains largely unknown. One approach combines the specificity of monoclonal antibodies, which recognize a three-dimensional epitope, with ligand binding. Using purified retinoic acid receptor gamma D and E domains, a panel of six unique monoclonal antibodies were isolated and characterized using solid-state receptor binding and retinoic acid receptor (RAR)-RXR heterodimer supershift formation. Three antibodies are specific for RARgamma (mAbI, mAbII, and mAbV) and four recognize a three-dimensional epitope (mAbI, mAbIV, mAbV, and mAbVI). Three antibodies (mAbIII, mAbV, and mAbVI) dissociate from the receptor in electrophoretic mobility shift assays upon the addition of retinoic acid. In particular, the binding characteristics of mAbIII, whose epitope was mapped to a region identified as an omega-loop (amino acids 207-222), suggest a model for ligand binding to the receptor. In this model, ligand binding causes a positioning of helix 12 into a favorable conformation for interaction with the transcriptional machinery. The Omega-loop then closes in order to stabilize this "active" position. The results reported here also suggest that a region of the hinge or D domain of the receptor (amino acids 156-188), an area that can play a role in protein-protein interactions, may also be important in ligand-induced functional changes.

Retinoic acid exerts its many biological effects by interaction with a nuclear protein, the retinoic acid receptor (RAR). The details of this interaction are unknown due mainly to the lack of sufficient quantities of pure functional receptor protein for biochemical and structural studies. We have recently subcloned the D and E domains of human RAR gamma for expression in Escherichia coli. Using nickel-chelation affinity chromatography with a polyhistidine amino-terminal tail, purification of the DE peptide with a pI of 5.18 was accomplished to greater than 98% purity. Scatchard analysis and fluorescence quenching techniques using the purified protein indicate a very high percentage of functional molecules ( > 95%) with a Kd for retinoic acid (t-RA) of 0.6 +/- 0.1 nM. Circular dichroism spectra of the purified domains predict a predominantly alpha-helical structure (approximately 56%) with little beta sheet present. No significant changes in these structural characteristics were observed upon binding of t-RA. Inspection of the amino acid sequence within these domains identified a single tryptophan residue at position 227. Modeling the amino acid sequence in this region as an alpha-helical structure indicates that this tryptophan is adjacent to alanine 234, which corresponds to alanine 225 in RAR beta that has previously been linked to the ligand binding site. Fluorescence of this tryptophan was quenched in a dose-dependent manner on the addition of t-RA, confirming that Trp-227 is within the ligand binding site. Tryptophan fluorescence quenching analysis also demonstrates that a single retinoic acid molecule is bound per receptor and suggests that receptor-ligand interactions occur within the amino-terminal portion of the predominantly alpha-helical ligand binding domain.

The yeast translational elongation factor 3 (EF-3) stimulates EF-1 alpha-dependent binding of aminoacyl-tRNA by the ribosome. The requirement for EF-3 is unique to fungi; a functional analog has not been found in prokaryotes or other eukaryotes. We have isolated and characterized the structural gene, YEF3, that encodes EF-3. The YEF3 gene is present in one copy/haploid genome and is essential for vegetative growth. DNA sequence analysis revealed that the YEF3 gene contains an open reading frame of 1044 codons. The deduced amino acid sequence contains two repeats of a nucleotide-binding motif, which is similar to the nucleotide-binding consensus sequences of hydrophilic, membrane-associated ATPases. EF-3 catalyzes ATP hydrolysis in a ribosome-dependent manner. A modified assay procedure has been developed that allows measurement of the ATP hydrolytic activity of EF-3 in cell-free extracts without interference by other nucleotide hydrolyase activities. Using this modified assay, we have shown that the wild-type YEF3 gene restores heat stable EF-3 activity in a yeast strain containing a temperature-sensitive EF-3. Introduction of the YEF3 gene on a high copy number plasmid into yeast strains increases the ribosome-dependent ATPase activity. The level of EF-3 protein is also increased 3-5-fold. Elevated EF-3 protein levels did not cause a significant increase in EF-1 alpha and EF-2 protein. Yeast strains containing elevated EF-3 protein levels are more sensitive to the aminoglycoside antibiotics hygromycin and paromomycin. These drugs are known to increase translational errors. This observation suggests that EF-3 may indirectly affect translational accuracy.