Murine Leukemia Virus Pseudoknot: Structural Analysis of Conformational Changes

The Murine Leukemia Virus’ (MLV) pseudoknot is an RNA motif that regulates readtrough translation, which is critical for packaging and maturation of new viral particles. The MLV pseudoknot undergoes a p.H dependent conformational change to a structure that regulates the readtrough mechanism of the retrovirus. However, at low pH NMR signals are too broad due to the fast exchange between the two conformers, and the structural analysis of the triple helix conformation is ambiguous. We postulate that design of a construct that trapped into a single folded conformation will help determine the structure of the triple helix conformation using NMR. In order to test our hypothesis, we analyzed 3 constructs. The first construct has a deleted GC base pair on stem 2 creating an artificial bend of stem. This construct cannot be tested by in vitro translational assays, so a double mutation –GC and replacement of Adenine 38 for Uracil (U38A‐GC) was also designed. In the last construct, Adenine 17 was replaced by a Uracil (A17U) eliminating the protonation of stem 2. Our results indicate that –GC base pair construct has lost pH dependence, and it is in slow exchange between two conformations; spectra of U38A‐GC construct exhibits characteristics of the native pseudoknot at low pH. Finally, spectra from construct A17U indicate that the triple helix is still pH dependant suggesting the need of multiple protonation events.