Formation of frameshift-stimulating RNA pseudoknots is facilitated by remodeling of their folding intermediates.

Programmed -1 ribosomal frameshifting is an essential regulation mechanism of translation in viruses and bacteria. It is stimulated by mRNA structures inside the coding region. As the structure is unfolded repeatedly by consecutive translating ribosomes, whether it can refold properly each time is important in performing its function. By using single-molecule approaches and molecular dynamics simulations, we found that a frameshift-stimulating RNA pseudoknot folds sequentially through its upstream stem S1 and downstream stem S2. In this pathway, S2 folds from the downstream side and tends to be trapped in intermediates. By masking the last few nucleotides to mimic their gradual emergence from translating ribosomes, S2 can be directed to fold from the upstream region. The results show that the intermediates are greatly suppressed, suggesting that mRNA refolding may be modulated by ribosomes. Moreover, masking the first few nucleotides of S1 favors the folding from S2 and yields native pseudoknots, which are stable enough to retrieve the masked nucleotides. We hypothesize that translating ribosomes can remodel an intermediate mRNA structure into a stable conformation, which may in turn stimulate backward slippage of the ribosome. This supports an interactive model of ribosomal frameshifting and gives an insightful account addressing previous experimental observations.

The metabolic role of human ADH3 functioning as ethanol dehydrogenase.

Human class III alcohol dehydrogenase (ADH3), also known as glutathione-dependent formaldehyde dehydrogenase, exhibited non-hyperbolic kinetics with ethanol at a near physiological pH 7.5. The S(0.5) and k(cat) were determined to be 3.4+/-0.3 M and 33+/-3 min(-1), and the Hill coefficient (h) 2.21+/-0.09, indicating positive cooperativity. Strikingly, the S(0.5) for ethanol was found to be 5.4 x 10(6)-fold higher than the K(m) for S-(hydroxymethyl)glutathione, a classic substrate for the enzyme, whereas the k(cat) for the former was 41% lower than that for the latter. Isotope effects on enzyme activity suggest that hydride transfer may be rate-limiting in the oxidation of ethanol. Kinetic simulations using the experimentally determined Hill constant suggest that gastric ADH3 may highly effectively contribute to the first-pass metabolism at 0.5-3 M ethanol, an attainable range in the gastric lumen during alcohol consumption. The positive cooperativity mainly accounts for this metabolic role of ADH3.