Comparative studies of frameshifting and nonframeshifting RNA pseudoknots: A mutational and NMR investigation of pseudoknots derived from the bacteriophage T2 gene 32 mRNA and the retroviral gag-pro frameshift site

Abstract

Mutational and NMR methods were used to investigate features of sequence, structure, and dynamics that are associated with the ability of a pseudoknot to stimulate a −1 frameshift. In vitro frameshift assays were performed on retroviral gag-pro frameshift-stimulating pseudoknots and their derivatives, a pseudoknot from the gene 32 mRNA of bacteriophage T2 that is not naturally associated with frameshifting, and hybrids of these pseudoknots. Results show that the gag-pro pseudoknot from human endogenous retrovirus-K10 (HERV) stimulates a −1 frameshift with an efficiency similar to that of the closely related retrovirus MMTV. The bacteriophage T2 mRNA pseudoknot was found to be a poor stimulator of frameshifting, supporting a hypothesis that the retroviral pseudoknots have distinctive properties that make them efficient frameshift stimulators. A hybrid, designed by combining features of the bacteriophage and retroviral pseudoknots, was found to stimulate frameshifting while retaining significant structural similarity to the nonframeshifting bacteriophage pseudoknot. Mutational analyses of the retroviral and hybrid pseudoknots were used to evaluate the effects of an unpaired (wedged) adenosine at the junction of the pseudoknot stems, changing the base pairs near the junction of the two stems, and changing the identity of the loop 2 nucleotide nearest the junction of the stems. Pseudoknots both with and without the wedged adenosine can stimulate frameshifting, though the identities of the nucleotides near the stem1/stem2 junction do influence efficiency. NMR data showed that the bacteriophage and hybrid pseudoknots are similar in their local structure at the junction of the stems, indicating that pseudoknots that are similar in this structural feature can differ radically in their ability to stimulate frameshifting. NMR methods were used to compare the internal motions of the bacteriophage T2 pseudoknot and representative frameshifting pseudoknots. The stems of the investigated pseudoknots are similarly well ordered on the time scales to which nitrogen-15 relaxation data are sensitive; however, solvent exchange rates for protons at the junction of the two stems of the nonframeshifting bacteriophage pseudoknot are significantly slower than the analogous protons in the representative frameshifting pseudoknots.