An intermolecular RNA triplex provides insight into structural determinants for the pseudoknot stimulator of −1 ribosomal frameshifting

Abstract

An efficient −1 programmed ribosomal frameshifting (PRF) signal requires an RNA slippery sequence and a downstream RNA stimulator, and the hairpin-type pseudoknot is the most common stimulator. However, a pseudoknot is not sufficient to promote −1 PRF. hTPK-DU177, a pseudoknot derived from human telomerase RNA, shares structural similarities with several −1 PRF pseudoknots and is used to dissect the roles of distinct structural features in the stimulator of −1 PRF. Structure-based mutagenesis on hTPK-DU177 reveals that the −1 PRF efficiency of this stimulator can be modulated by sequential removal of base–triple interactions surrounding the helical junction. Further analysis of the junction-flanking base triples indicates that specific stem–loop interactions and their relative positions to the helical junction play crucial roles for the −1 PRF activity of this pseudoknot. Intriguingly, a bimolecular pseudoknot approach based on hTPK-DU177 reveals that continuing triplex structure spanning the helical junction, lacking one of the loop-closure features embedded in pseudoknot topology, can stimulate −1 PRF. Therefore, the triplex structure is an essential determinant for the DU177 pseudoknot to stimulate −1 PRF. Furthermore, it suggests that −1 PRF, induced by an in-trans RNA via specific base–triple interactions with messenger RNAs, can be a plausible regulatory function for non-coding RNAs.

Functional analysis of the SRV-1 RNA frameshifting pseudoknot

“…This pseudoknot has recently been shown to function in frameshifting in vitro (22). Also here the adenosines formed triple interactions with base pairs in stem S1 and were essential for frameshifting (22,34). The effect of substituting the second C in L2 has not been investigated; its presence may be required to prevent slippage of the RNA polymerase.…”

Secondary structure of bacteriophage T4 gene 60 mRNA: Implications for translational bypassing

“…Highly structured segments, such as pseudoknots and stable hairpins, have been shown to modulate the movement of the ribosome along an mRNA inducing shifts in reading frame (Farabaugh 1996;Herr et al 2000a;Kontos et al 2001;Giedroc and Cornish 2009;Mazauric et al 2009;Chou and Chang 2010;Chung et al 2010), presumably due to the difficulty that the ribosome has unwinding complex, stable structures (Firth and Brierley 2012). In these cases, multiple protein products are encoded in overlapping reading frames, and the efficiency of the frameshifting event determines the relative ratio at which each protein is expressed (Farabaugh 1996;Chou and Chang 2010).…”

“…In these cases, multiple protein products are encoded in overlapping reading frames, and the efficiency of the frameshifting event determines the relative ratio at which each protein is expressed (Farabaugh 1996;Chou and Chang 2010). In these ways, mRNA provides additional layers of regulation for gene expression beyond those at the DNA and protein levels (Herr et al 2000a;Kontos et al 2001).…”

Augmented genetic decoding: global, local and temporal alterations of decoding processes and codon meaning

“…Nascent peptides are also known to modulate ribosomal frameshifting 121,122 . The largest class of stimulators are RNA secondary structures: stem-loops [123][124][125] , simple 126 and relatively complex RNA pseudoknots containing extra stems [127][128][129] , or triple helices 130,131 , kissing loops 132 , G-quadruplexes 133,134 or long-range interactions 135,136 (see REFS 22,[137][138][139] for reviews). mRNA interacts with various cellular components, and these interactions may alter the stimulatory properties of particular structures or sequences.…”