Structural and functional studies of retroviral RNA pseudoknots involved in ribosomal frameshifting: nucleotides at the junction of the two stems are important for efficient ribosomal frameshifting.

Abstract

Ribosomal frameshifting, a translational mechanism used during retroviral replication, involves a directed change in reading frame at a specific site at a defined frequency. Such programmed frameshifting at the mouse mammary tumor virus (MMTV) gag‐pro shift site requires two mRNA signals: a heptanucleotide shifty sequence and a pseudoknot structure positioned downstream. Using in vitro translation assays and enzymatic and chemical probes for RNA structure, we have defined features of the pseudoknot that promote efficient frameshifting. Heterologous RNA structures, e.g. a hairpin, a tRNA or a synthetic pseudoknot, substituted downstream of the shifty site fail to promote frameshifting, suggesting that specific features of the MMTV pseudoknot are important for function. Site‐directed mutations of the MMTV pseudoknot indicate that the pseudoknot junction, including an unpaired adenine nucleotide between the two stems, provides a specific structural determinant for efficient frameshifting. Pseudoknots derived from other retroviruses (i.e. the feline immunodeficiency virus and the simian retrovirus type 1) also promote frameshifting at the MMTV gag‐pro shift site, dependent on the same structure at the junction of the two stems.

A loop 2 cytidine-stem 1 minor groove interaction as a positive determinant for pseudoknot-stimulated –1 ribosomal frameshifting

“…Our data are consistent with a scenario where a local destabilization allows the elongating ribosome to unfold the distal pseudoknot-forming stem S2 before significant partitioning into the Ϫ1 reading frame. In fact, although the data are limited, the destabilization separating wild-type and C27A ScYLV RNAs is similar in magnitude to the degree to which the helical junction regions are destabilized in other frameshifting systems (⌬⌬G 37 Ϸ 1-2 kcal⅐mol Ϫ1 ) due to substitutions or deletions of junction nucleotides that negatively impact frameshift stimulation (24,28,29,40,48,49). Indeed, if the entire ground-state destabilization of mutant pseudoknots observed here (⌬⌬G 37 Ϸ 1.5 kcal⅐mol Ϫ1 ) is manifest as a decrease in the energy barrier to ribosome-promoted pseudoknot unwinding, ⌬⌬G ‡ , then this would translate into an Ϸ10-fold increase in the rate of unfolding in destabilized pseudoknots (14).…”

Mechanical Unfolding of the Beet Western Yellow Virus −1 Frameshift Signal

“…The ratio of the two reporter proteins can be compared to determine the frequency of a frameshifting event. Frameshifting efficiency varies widely among pseudoknots, and altering the sequence of the pseudoknot can substantially affect the efficiency (24).…”

“…Studies have found that thermodynamic stability of the pseudoknot is not correlated with frameshifting efficiency (24). That is, frameshifting and nonframeshifting structures have similar free energies.…”

“…Recent single-molecule unfolding of the Infectious Bronchitis Virus (IBV) pseudoknot reveal a connection between higher unfolding forces and higher frameshifting efficiencies, and a reverse correlation was observed between the rate of mechanical unfolding and frameshifting efficiency in a specific narrow range of forces for similar pseudoknots (26,72). Furthermore, a simple theoretical model of frameshifting has yielded a correlation between frameshift efficiencies and unfolding force rather than energy (24).…”

A Local Structural Prediction Algorithm for RNA Triple Helix Structure

“…Triple helix structures exist in yeast and human telomerase, and are found to be essential in quite a few biological processes (e.g. chromosome stability in stem cells, germline cells and cancer cells [8][9][10]; ribosomal frameshifting [11,12]). …”