Deconstruction of GCN4/GCRE into a monomeric peptide-DNA complex

Here we describe a system that enables short peptides to bind DNA sequence-specifically. Linking the peptide covalently to DNA through a disulphide bond eliminates the unfavourable energetic cost of diffusion and thus potentiates the peptide-DNA interaction. By this approach we have deconstructed the GCN4/DNA complex into its elemental DNA recognition units. We find that the GCN4 basic region contacts the two half-sites with very different affinities and propose that this thermodynamic asymmetry plays a role in differential regulation of gene expression. Specific binding of the peptide to DNA stabilizes the disulphide bond toward reduction suggesting a novel approach to the discovery of new DNA-binding specificities.     

Subdomain folding of the coiled coil leucine zipper from the bZIP transcriptional activator GCN4

One popular model for protein folding, the framework model, postulates initial formation of secondary structure elements, which then assemble into the native conformation. However, short peptides that correspond to secondary structure elements in proteins are often only marginally stable in isolation. A 33-residue peptide (GCN4-p1) corresponding to the GCN4 leucine zipper folds as a parallel, two-stranded coiled coil [O'Shea, E.K., Klemm, J.D., Kim, P.S., & Alber, T.A. (1991) Science 254, 539-544]. Deletion of the first residue (Arg 1) results in local, N-terminal unfolding of the coiled coil, suggesting that a stable subdomain of GCN4-p1 can form. N- and C-terminal deletion studies result in a 23-residue peptide, corresponding to residues 8-30 of GCN4-p1, that folds as a parallel, two-stranded coil with substantial stability (the melting temperature of a 1 mM solution is 43 degrees C at pH 7). In contrast, a closely related 23-residue peptide (residues 11-33 of GCN4-p1) is predominantly unfolded, even at 0 degrees C, as observed previously for many isolated peptides of similar length. Thus, specific tertiary packing interactions between two short units of secondary structure can be energetically more important in stabilizing folded structure than secondary structure propensities. These results provide strong support for the notion that stable, cooperatively folded subdomains are the important determinants of protein folding.     

Using GCN4 as a reporter of eIF2 alpha phosphorylation and translational regulation in yeast

Molecular genetic analyses in yeast are a powerful method to study gene regulation. Conservation of the mechanism and regulation of protein synthesis between yeast and mammalian cells makes yeast a good model system for the analysis of translation. One of the most common mechanisms of translational regulation in mammalian cells is the phosphorylation of serine-51 on the alpha subunit of the translation initiation factor elF2, which causes an inhibition of general translation. In contrast, in the yeast Saccharomyces cerevisiae phosphorylation of elF2 alpha on serine-51 by the GCN2 protein kinase mediates the translational induction of GCN4 expression. The unique structure of the GCN4 mRNA makes GCN4 expression especially sensitive to elF2 alpha phosphorylation, and the simple microbiological tests developed in yeast to analyze GCN4 expression serve as good reporters of elF2 alpha phosphorylation. It is relatively simple to express heterologous proteins in yeast, and it has been shown that the mammalian elF2 alpha kinases will functionally substitute for GCN2. Structure-function analyses of translation factors or translational regulators can also be performed by assaying for effects on general and GCN4-specific translation. Three tests can be used to study elF2 alpha phosphorylation and/or translational activity in yeast. First, general translation can be monitored by simple growth tests, while GCN4 expression can be analyzed using sensitive replicaplating tests. Second, GCN4 translation can be quantitated by measuring expression from GCN4-lacZ reporter constructs. Finally, isoelectric focusing gels can be used to directly monitor in vivo phosphorylation of elF2 alpha in yeast.     

Thermodynamics of extraction of Re O 4 − from aqueous sulfuric acid media with Tri-n-butyl phosphate dissolved in kerosene

Rhenium distribution between Tri-n-butyl phosphate (TBP) dissolved in kerosene and sulfuric aqueous media is investigated under different temperatures, acidities, and TBP concentrations. Results show an exothermic extraction reaction with a distribution factor that decreases with the increase of the temperature and increases with the increase of the acidity and the TBP concentration. The stoichiometry of the reaction indicates neutralization of 1 mole of H⁺ with 1 mole of ReO ₄ ⁻ and solvating of a neutral molecule with four molecules of TBP. The structure of the complexes formed is, therefore, 4TBP · HReO₄. The apparent Gibbs free energy of the formation of 4TBP · HReO₄ from TBP, ReO ₄ ⁻ , and H⁺ is determined to be equal to −66,989 + 219.8 T J/mole.