Recent advances in our understanding of translational dynamics indicate that codon usage and mRNA secondary structure influence translation and protein folding. The most frequent cause of cystic fibrosis (CF) is the deletion of three nucleotides (CTT) from the cystic fibrosis transmembrane conductance regulator (CFTR) gene that includes the last cytosine (C) of isoleucine 507 (Ile507ATC) and the two thymidines (T) of phenylalanine 508 (Phe508TTT) codons. The consequences of the deletion are the loss of phenylalanine at the 508 position of the CFTR protein (⌬F508), a synonymous codon change for isoleucine 507 (Ile507ATT), and protein misfolding. Here we demonstrate that the ⌬F508 mutation alters the secondary structure of the CFTR mRNA. Molecular modeling predicts and RNase assays support the presence of two enlarged single stranded loops in the ⌬F508 CFTR mRNA in the vicinity of the mutation. The consequence of ⌬F508 CFTR mRNA "misfolding" is decreased translational rate. A synonymous single nucleotide variant of the ⌬F508 CFTR (Ile507ATC), that could exist naturally if Phe-508 was encoded by TTC, has wild type-like mRNA structure, and enhanced expression levels when compared with native ⌬F508 CFTR. Because CFTR folding is predominantly cotranslational, changes in translational dynamics may promote ⌬F508 CFTR misfolding. Therefore, we propose that mRNA "misfolding" contributes to ⌬F508 CFTR protein misfolding and consequently to the severity of the human ⌬F508 phenotype. Our studies suggest that in addition to modifier genes, SNPs may also contribute to the differences observed in the symptoms of various ⌬F508 homozygous CF patients.

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The peroxy isomers of nitrate and nitric acid, peroxynitrite and peroxynitrous acid, are studied with ab initio and density functional methods. The results are compared to the observed Raman and 15N NMR spectra. The harmonic vibrational frequencies, NMR chemical shifts, and energies clearly favor cis ONOO- as the most stable and predominant ONOO- isomer. Peroxynitrite has a large rotational barrier of ∼24 kcal/mol because of partial π-bonding in the central bond. This is confirmed by a bond order of 1.5 for cis and trans ONOO- computed by electron density analysis. Electron correlation is critical in accurately predicting the relative energies for this system, as Hartree−Fock predicts a lower triplet state. The intense, broad band in the solution Raman spectrum centered at 642 cm-1 is predicted to be too low by 100−150 cm-1 if the vibration is the cis torsion. Resolution of this discrepancy is attempted by estimating the effects of solvent and anharmonicity. The results on ONOO- are compared to those for ONOOH, which is adequately described by lower levels of theory. The planar cis−cis isomer of ONOOH is the lowest energy structure by 1−2 kcal/mol.