An Equilibrium-Dependent Retroviral mRNA Switch Regulates Translational Recoding

Most retroviruses require translational recoding of a viral messenger RNA stop codon to maintain a precise ratio of structural (Gag) and enzymatic (Pol) proteins during virus assembly. Pol is expressed exclusively as a Gag–Pol fusion either by ribosomal frameshifting or by read-through of the gag stop codon. Both of these mechanisms occur infrequently and only affect 5–10% of translating ribosomes, allowing the virus to maintain the critical Gag to Gag–Pol ratio. Although it is understood that the frequency of the recoding event is regulated by cis RNA motifs, no mechanistic explanation is currently available for how the critical protein ratio is maintained. Here we present the NMR structure of the murine leukaemia virus recoding signal and show that a protonation-dependent switch occurs to induce the active conformation. The equilibrium is such that at physiological pH the active, read-through permissive conformation is populated at approximately 6%: a level that correlates with in vivo protein quantities. The RNA functions by a highly sensitive, chemo-mechanical coupling tuned to ensure an optimal read-through frequency. Similar observations for a frameshifting signal indicate that this novel equilibrium-based mechanism may have a general role in translational recoding.Molecular and Cellular Biolog

Fluorescence Competition Assay Measurements of Free Energy Changes for RNA Pseudoknots

RNA pseudoknots have important functions, and thermodynamic stability is a key to predicting pseudoknots in RNA sequences and to understanding their functions. Traditional methods, such as UV melting and differential scanning calorimetry, for measuring RNA thermodynamics are restricted to temperature ranges around the melting temperature for a pseudoknot. Here, we report RNA pseudoknot free energy changes at 37 °C measured by fluorescence competition assays. Sequence-dependent studies for the loop 1−stem 2 region reveal (1) the individual nearest-neighbor hydrogen bonding (INN-HB) model provides a reasonable estimate for the free energy change when a Watson−Crick base pair in stem 2 is changed, (2) the loop entropy can be estimated by a statistical polymer model, although some penalty for certain loop sequences is necessary, and (3) tertiary interactions can significantly stabilize pseudoknots and extending the length of stem 2 may alter tertiary interactions such that the INN-HB model does not predict the net effect of adding a base pair. The results can inform writing of algorithms for predicting and/or designing RNA secondary structures

Conformational study of peptides containing dehydrophenylalanine Helical structures without hydrogen bond

417-425The conformational behaviour of ∆zPhe has been investigated in the model dipeptide Ac-∆z Phe-NHMe and in the model tripeptides Ac-X-∆z Phe-NHMe with X=Gly, Ala,Val, Leu, Abu, Aib and Phe and is found to be quite different. In the model tripeptides with X=Ala, Val, Leu, Abu, Phe the most stable structure corresponds to 1=-30°, ψ1= 120° and 2=ψ2 =30°. This structure is stabilized by the hydrogen bond formation between C=O of acetyl group and the NH of the amide group, resulting in the formation of a 10-membered ring but not a 310 helical structure. In the peptides Ac- Aib-∆z Phe-NHMe and Ac-(Aib-∆z Phe)3-NHMe, the helical conformers with =±30°, <span style="font-size:14.0pt;mso-bidi-font-size: 12.0pt" lang="EN-IN">ψ= ±60° for Aib residue and = ψ= ±30° for ∆z Phe are predicted to be most stable. The computational studies for the positional preferences of ∆z Phe residue in the peptide containing one ∆z Phe and nine Ala residues reveal the formation of a 310 helical structure in all the cases with terminal preferences for ∆zPhe.  The conformational behaviour of Ac-(∆z Phe)n-NHMe with n≤4 is predicted to bc very labile. With n> 4, degenerate conformational states with  <span style="font-size:16.0pt; font-family:Symbol;mso-ascii-font-family:" arial="" unicode="" ms";mso-hansi-font-family:="" "arial="" ms";mso-char-type:symbol;mso-symbol-font-family:symbol"="" lang="EN-IN">j, <span style="font-size:14.0pt;mso-bidi-font-size: 12.0pt" lang="EN-IN">ψ values of 0° ± 90° adopt helical structures which are stabilized by carbonyl-carbonyl interactions and the N-H-π interactions between the amino group of every ∆z Phe residue with one C-C edge of its own phenyl ring. The results are in agreement with the experimental finding that screw sense of helix for peptides containing ∆z Phe residues is ambiguous in solution. The helical structures stabilized by hydrogen bond formation are found to be at least 3kCalmol-1 less stable. Conformational studies have also been carried out for the peptide Ac-(∆E Phe)6,-NHMe and the peptide Ac-∆Ala- (∆z Phe)6-NHMe containing ∆Ala residue at the N-terminal. The N-H -π interactions are absent in peptide Ac-(∆E Phe)6-NHMe. </span