The role of RNA pseudoknot stem 1 length in the promotion of efficient −1 ribosomal frameshifting

Abstract

The ribosomal frameshifting signal present in the genomic RNA of the coronavirus infectious bronchitis virus (IBV) contains a classic hairpin-type RNA pseudoknot that is believed to possess coaxially stacked stems of 11 bp (stem 1) and 6 bp (stem 2). We investigated the influence of stem 1 length on the frameshift process by measuring the frameshift efficiency in vitro of a series of IBV-based pseudoknots whose stem 1 length was varied from 4 to 13 bp in single base-pair increments. Efficient frameshifting depended upon the presence of a minimum of 11 bp; pseudoknots with a shorter stem 1 were either non-functional or had reduced frameshift efficiency, despite the fact that a number of them had a stem 1 with a predicted stability equal to or greater than that of the wild-type IBV pseudoknot. An upper limit for stem 1 length was not determined, but pseudoknots containing a 12 or 13 bp stem 1 were fully functional. Structure probing analysis was carried out on RNAs containing either a ten or 11 bp stem 1; these experiments confirmed that both RNAs formed pseudoknots and appeared to be indistinguishable in conformation. Thus the difference in frameshifting efficiency seen with the two structures was not simply due to an inability of the 10 bp stem 1 construct to fold into a pseudoknot. In an attempt to identify other parameters which could account for the poor functionality of the shorter stem 1-containing pseudoknots, we investigated, in the context of the 10 bp stem 1 construct, the influence on frameshifting of altering the slippery sequence-pseudoknot spacing distance, loop 2 length, and the number of G residues at the bottom of the 5'-arm of stem 1. For each parameter, it was possible to find a condition where a modest stimulation of frameshifting was observable (about twofold, from seven to a maximal 17 %), but we were unable to find a situation where frameshifting approached the levels seen with 11 bp stem 1 constructs (48-57 %). Furthermore, in the next smaller construct (9 bp stem 1), changing the bottom four base-pairs to G.C (the optimal base composition) only stimulated frameshifting from 3 to 6 %, an efficiency about tenfold lower than seen with the 11 bp construct. Thus stem 1 length is a major factor in determining the functionality of this class of pseudoknot and this has implications for models of the frameshift process.

The 9-Å solution: How mRNA pseudoknots promote efficient programmed −1 ribosomal frameshifting

“…This in turn would impact on frameshifting efficiencies. Direct experiments to test this are consistent with this prediction (Napthine et al 1999), as are results obtained with residue swapping mutants at the base of stem 1 (Kim et al 1999;Nixon et al 2002). Eukaryotic ribosomes also contain homologs for the three bacterial proteins mapped to the entrance of the mRNA tunnel.…”

Contribution of the intercalated adenosine at the helical junction to the stability of the gag-pro frameshifting pseudoknot from mouse mammary tumor virus

“…Precisely how RNA pseudoknots positioned downstream of a slippery site stimulate Ϫ1 ribosomal frameshifting is currently unknown, but may involve features of a particular structure, and/or tertiary folds of an appropriate stability+ Structure-probing and NMR data currently available for pseudoknots derived from retroviruses, coronaviruses, and luteoviruses, however, suggest that there may well be more than one structural motif that can stimulate ribosomal frameshifting Alam et al+, 1999;Napthine et al+, 1999)+ However, with respect to MMTV and closely related viruses, a bent conformation facilitated by the wedged adenosine appears to be a primary determinant for frameshifting (Chen et al+, 1995(Chen et al+, , 1996Sung & Kang, 1998)+…”

“…The work presented here shows that pseudoknots that strongly stimulate frameshifting, MMTV vpk and U13C RNAs, and a coaxially stacked RNA that shows little stimulation of frameshifting, U13C⌬A14, all form pseudoknots of very similar global stabilities and mono-, di-, and multivalent ion-binding properties+ We could find no evidence for differential accumulation of monovalent or divalent ions in the coaxially stacked versus bent pseudoknots or for significant stabilizing interactions in loop 2 that are dependent on specific coordination of monovalent ions in the context of wedged versus ⌬A14 pseudoknots+ All RNAs studied here appear to unfold via the same hairpin intermediate, which is separated in free energy from the pseudoknot by relatively similar extents in all three cases, with the wedged and ⌬A14 pseudoknots differing from each other by 1+4 kcal mol Ϫ1 at 1 M Na ϩ + Although it is unknown to what extent such a difference in stabilization might alter the population distribution of RNA structures (folded and unfolded) at the ribosome, it seems unlikely that frameshifting efficiency will be strongly correlated with the global stability of the RNAs+ This is consistent with previous findings that show that insertion of an RNA hairpin of a stability equal to or greater than that predicted for the pseudoknot is far less effective at stimulating frameshifting than is the pseudoknot itself (Brierley et al+, 1991;ten Dam et al+, 1995)+ Apart from global structure and stability, another feature that might be exploited by the ribosome is the rate at which the fully folded and partially folded conformers can interconvert+ It may be possible that insertion of a wedged nucleotide and/or the presence of other stabilizing interactions at or near the helical junction are particularly poised to alter the rate of pseudoknot unfolding, even in the absence of relatively small differences in global stability+ Certainly, recent work derived from analyzing the effects of loop 2-stem 1 compensatory mutations on frameshifting efficiency of MMTV-IBV chimeric pseudoknots supports the presence of defined, though not yet structurally characterized, interactions between the 39 nucleotide in loop 2 (an adenosine) and the base of stem 1 nearest the helical junction, which are functionally important in some contexts (Liphardt et al+, 1999)+ The extent to which these interactions contribute to the stability is unknown, but it is likely to be small compared to the global stability of the RNA if our work on the mIAP (Theimer & Giedroc, 1999) and MMTV pseudoknots (this work) is generalizable to other gag-pro frameshifters+ It may be that these interactions provide a topological constraint to pseudoknot unwinding or otherwise subtly alter the junction flexibility in such a way that is compatible with function+ A major finding is that we have quantified the thermodynamic effects of the deletion of an unpaired adenosine nucleotide on the stability of an H-type RNA pseudoknot involved in ribosomal frameshifting+ We find that this deletion is modestly destabilizing by 1+4 kcal mol Ϫ1 + This unpaired adenosine provides a stabilizing free energy increment similar in magnitude to that of an 39 unpaired nucleotide stacked on a closing base pair at a helical terminus+ Knowledge of this free energy increment may further enable prediction of RNA secondary structure from nucleotide sequence+…”

“…The mouse mammary tumor virus (MMTV) gag-pro 1 frameshifting pseudoknot has been subjected to mutational analysis and structure probing (Chamorro et al+, 1992;Chen et al+, 1995) and the solution structure has been solved by NMR spectroscopy + Recently, a refined NMR solution structure of the MMTV pseudoknot was published with a larger number of loop-to-stem constraints for loop 1 because of NOEs with a coordinated Co(NH 3 ) 6 3ϩ ion (Gonzalez & Tinoco, 1999)+ Solution structures of frameshifting and nonframeshifting variants have also been determined (Chen et al+, 1996;Kang et al+, 1996;Kang & Tinoco, 1997)+ Frameshifting efficiency in these MMTV pseudoknot derivatives was found to correlate with a specific bent conformation where the helical junction was prevented from coaxially stacking by a single unpaired adenosine intercalated between the two stems+ Further evidence for the importance of an unpaired nucleotide(s) at the helical junction comes from functional studies on pseudoknot chimeras formed between the infectious bronchitis virus (IBV)1a-1b and MMTV gagpro pseudoknots (Liphardt et al+, 1999), in which high efficiency frameshifting RNAs were found to contain an unpaired adenosine at the junction of two six-base pair stems in addition to other favorable and functionally important interactions between the 39 end of loop 2 and the base pairs at the bottom of stem 1 proximal to the junction+ The extent to which these structural determinants characterize other gag-pol pseudoknots, like simian retrovirus-1 (SRV-1) and feline immunodeficiency virus (FIV) is not known, although a weakly basepaired or unpaired junction may characterize these systems as well (Morikawa & Bishop, 1992;Chen et al+, 1995;Sung & Kang, 1998)+ Folding studies on variants of the gag-pro frameshifting pseudoknot from mouse intracisternal A-type particles (mIAP), reveal that this RNA has a weakly paired or nonbasepaired junction similar to that of SRV-1, because the small destabilization observed upon deletion of the helical junction adenosine [⌬⌬G8 37 ' 1 kcal mol Ϫ1 ] could not be attributed to the loss of an A-U base pair (Theimer & Giedroc, 1999)+ Studies on the thermodynamics of folding of frameshifting versus nonframeshifting pseudoknots are required to determine the origin of the stability of active versus inactive conformations as well as the degree to which active conformers are stabilized relative to nonframeshifting or partially folded molecules+ In this study, we have examined the thermodynamic and ion-binding properties of frameshifting and nonframeshifting variants of the MMTV gag-pro pseudoknot and have determined the energetics and contribution that an unpaired adenosine at the helical junction makes to the global stability of this pseudoknot+ This is an important issue since a single nucleotide bulge at the junction of two helical stems can strongly alter the stacking of the pseudoknot stems and, in turn, have unpredictable effects on the thermodynamic stability of the pseudoknot+ Indeed, in all cases that we have studied, loop substitution or deletion mutations are never energetically silent, and can have si...…”

Ribosomal Frameshift Signals in Viral Genomes

“…Additionally there is an example of a kissing stem-loop that promotes coronavirus frameshifting (Herold and Siddell, 1993). A lot of nuclease mapping and NMR data is available for some coronavirus signals but this does not reflect the diversity of nidovirus frameshift signals (Dos Ramos et al, 2004;Napthine et al, 1999;Plant et al, 2005Plant et al, , 2010Su et al, 2005).…”