Stem-loop structures can effectively substitute for an RNA pseudoknot in -1 ribosomal frameshifting

Programmed ribosomal frameshifting (PRF) is a conserved translational recoding mechanism found in all branches of life and viruses. In bacteria, archaea, and eukaryotes PRF is used to downregulate protein production by inducing a premature termination of translation, which triggers messenger RNA (mRNA) decay. In viruses, PRF is used to drive the production of a new protein while downregulating the production of another protein, thus maintaining a stoichiometry optimal for productive infection. Traditionally, PRF motifs have been defined by the characteristics of two cis elements: a slippery heptanucleotide sequence followed by an RNA pseudoknot or stem-loop within the mRNA. Recently, additional cis and new trans elements have been identified that regulate PRF in both host and viral translation. These additional factors suggest PRF is an evolutionarily conserved process whose function and regulation we are just beginning to understand.

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Structural dynamics of single SARS-CoV-2 pseudoknot molecules reveal topologically distinct conformers

Nature Communications ◽

10.1038/s41467-021-25085-6 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Krishna Neupane ◽

Meng Zhao ◽

Aaron Lyons ◽

Sneha Munshi ◽

Sandaru M. Ileperuma ◽

...

Keyword(s):

Optical Tweezers ◽

Structural Dynamics ◽

Drug Target ◽

The Other ◽

Conformational Heterogeneity ◽

Mechanical Tension ◽

Stem Loop ◽

Ribosomal Frameshifting ◽

Multiple Structures ◽

Rna Pseudoknot

AbstractThe RNA pseudoknot that stimulates programmed ribosomal frameshifting in SARS-CoV-2 is a possible drug target. To understand how it responds to mechanical tension applied by ribosomes, thought to play a key role during frameshifting, we probe its structural dynamics using optical tweezers. We find that it forms multiple structures: two pseudoknotted conformers with different stability and barriers, and alternative stem-loop structures. The pseudoknotted conformers have distinct topologies, one threading the 5′ end through a 3-helix junction to create a knot-like fold, the other with unthreaded 5′ end, consistent with structures observed via cryo-EM and simulations. Refolding of the pseudoknotted conformers starts with stem 1, followed by stem 3 and lastly stem 2; Mg2+ ions are not required, but increase pseudoknot mechanical rigidity and favor formation of the knot-like conformer. These results resolve the SARS-CoV-2 frameshift signal folding mechanism and highlight its conformational heterogeneity, with important implications for structure-based drug-discovery efforts.

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Structural dynamics of the SARS-CoV-2 frameshift-stimulatory pseudoknot reveal topologically distinct conformers

10.1101/2020.12.28.424630 ◽

2020 ◽

Author(s):

Krishna Neupane ◽

Meng Zhao ◽

Aaron Lyons ◽

Sneha Munshi ◽

Sandaru M Ileperuma ◽

...

Keyword(s):

Drug Discovery ◽

Optical Tweezers ◽

Structural Dynamics ◽

Drug Target ◽

Conformational Heterogeneity ◽

Computational Simulations ◽

Mechanical Tension ◽

Stem Loop ◽

Ribosomal Frameshifting ◽

Rna Pseudoknot

The RNA pseudoknot that stimulates −1 programmed ribosomal frameshifting in SARS coronavirus-2 (SARS-CoV-2) is a possible drug target. To understand how this 3-stemmed pseudoknot responds to the mechanical tension applied by ribosomes during translation, which is thought to play a key role during frameshifting, we probed its structural dynamics under tension using optical tweezers. Unfolding curves revealed that the frameshift signal formed multiple different structures: at least two distinct pseudoknotted conformers with different unfolding forces and energy barriers, as well as alternative stem-loop structures. Refolding curves showed that stem 1 formed first in the pseudoknotted conformers, followed by stem 3 and then stem 2. By extending the handle holding the RNA to occlude the 5′ end of stem 1, the proportion of the different pseudoknot conformers could be altered systematically, consistent with structures observed in cryo-EM images and computational simulations that had distinct topologies: the 5′ end of the RNA threaded through the 3-helix junction to form a ring-knot, or unthreaded as in more standard H-type pseudoknots. These results resolve the folding mechanism of the frameshift signal in SARS-CoV-2 and highlight the dynamic conformational heterogeneity of this RNA, with important implications for structure-based drug-discovery efforts.

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An RNA pseudoknot and an optimal heptameric shift site are required for highly efficient ribosomal frameshifting on a retroviral messenger RNA.

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.89.2.713 ◽

1992 ◽

Vol 89 (2) ◽

pp. 713-717 ◽

Cited By ~ 89

Author(s):

M. Chamorro ◽

N. Parkin ◽

H. E. Varmus

Keyword(s):

Messenger Rna ◽

Ribosomal Frameshifting ◽

Highly Efficient ◽

Rna Pseudoknot

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An atypical RNA pseudoknot stimulator and an upstream attenuation signal for -1 ribosomal frameshifting of SARS coronavirus

Nucleic Acids Research ◽

10.1093/nar/gki731 ◽

2005 ◽

Vol 33 (13) ◽

pp. 4265-4275 ◽

Cited By ~ 47

Author(s):

M.-C. Su

Keyword(s):

Sars Coronavirus ◽

Ribosomal Frameshifting ◽

Rna Pseudoknot

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Small synthetic molecule-stabilized RNA pseudoknot as an activator for –1 ribosomal frameshifting

Nucleic Acids Research ◽

10.1093/nar/gky689 ◽

2018 ◽

Vol 46 (16) ◽

pp. 8079-8089 ◽

Cited By ~ 8

Author(s):

Saki Matsumoto ◽

Neva Caliskan ◽

Marina V Rodnina ◽

Asako Murata ◽

Kazuhiko Nakatani

Keyword(s):

Ribosomal Frameshifting ◽

Rna Pseudoknot ◽

Synthetic Molecule

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Ribosomal pausing during translation of an RNA pseudoknot.

Molecular and Cellular Biology ◽

10.1128/mcb.13.11.6931 ◽

1993 ◽

Vol 13 (11) ◽

pp. 6931-6940 ◽

Cited By ~ 149

Author(s):

P Somogyi ◽

A J Jenner ◽

I Brierley ◽

S C Inglis

Keyword(s):

High Efficiency ◽

Mutational Analysis ◽

Base Pairs ◽

Stem Loop ◽

Pseudoknot Structure ◽

Stem Loop Structure ◽

Dead End ◽

Rna Pseudoknot ◽

Slippery Sequence

The genomic RNA of the coronavirus infectious bronchitis virus contains an efficient ribosomal frameshift signal which comprises a heptanucleotide slippery sequence followed by an RNA pseudoknot structure. The presence of the pseudoknot is essential for high-efficiency frameshifting, and it has been suggested that its function may be to slow or stall the ribosome in the vicinity of the slippery sequence. To test this possibility, we have studied translational elongation in vitro on mRNAs engineered to contain a well-defined pseudoknot-forming sequence. Insertion of the pseudoknot at a specific location within the influenza virus PB1 mRNA resulted in the production of a new translational intermediate corresponding to the size expected for ribosomal arrest at the pseudoknot. The appearance of this protein was transient, indicating that it was a true paused intermediate rather than a dead-end product, and mutational analysis confirmed that its appearance was dependent on the presence of a pseudoknot structure within the mRNA. These observations raise the possibility that a pause is required for the frameshift process. The extent of pausing at the pseudoknot was compared with that observed at a sequence designed to form a simple stem-loop structure with the same base pairs as the pseudoknot. This structure proved to be a less effective barrier to the elongating ribosome than the pseudoknot and in addition was unable to direct efficient ribosomal frameshifting, as would be expected if pausing plays an important role in frameshifting. However, the stem-loop was still able to induce significant pausing, and so this effect alone may be insufficient to account for the contribution of the pseudoknot to frameshifting.

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Ribosomal Pausing at a Frameshifter RNA Pseudoknot Is Sensitive to Reading Phase but Shows Little Correlation with Frameshift Efficiency

Molecular and Cellular Biology ◽

10.1128/mcb.21.24.8657-8670.2001 ◽

2001 ◽

Vol 21 (24) ◽

pp. 8657-8670 ◽

Cited By ~ 94

Author(s):

Harry Kontos ◽

Sawsan Napthine ◽

Ian Brierley

Keyword(s):

Rna Structure ◽

Phase Dependence ◽

Stem Loop ◽

Reading Frame ◽

Stem Loop Structure ◽

Rna Pseudoknot ◽

Ribosomal P ◽

Slippery Sequence ◽

The Impact ◽

Ribosomal Pausing

ABSTRACT Here we investigated ribosomal pausing at sites of programmed −1 ribosomal frameshifting, using translational elongation and ribosome heelprint assays. The site of pausing at the frameshift signal of infectious bronchitis virus (IBV) was determined and was consistent with an RNA pseudoknot-induced pause that placed the ribosomal P- and A-sites over the slippery sequence. Similarly, pausing at the simian retrovirus 1 gag/pol signal, which contains a different kind of frameshifter pseudoknot, also placed the ribosome over the slippery sequence, supporting a role for pausing in frameshifting. However, a simple correlation between pausing and frameshifting was lacking. Firstly, a stem-loop structure closely related to the IBV pseudoknot, although unable to stimulate efficient frameshifting, paused ribosomes to a similar extent and at the same place on the mRNA as a parental pseudoknot. Secondly, an identical pausing pattern was induced by two pseudoknots differing only by a single loop 2 nucleotide yet with different functionalities in frameshifting. The final observation arose from an assessment of the impact of reading phase on pausing. Given that ribosomes advance in triplet fashion, we tested whether the reading frame in which ribosomes encounter an RNA structure (the reading phase) would influence pausing. We found that the reading phase did influence pausing but unexpectedly, the mRNA with the pseudoknot in the phase which gave the least pausing was found to promote frameshifting more efficiently than the other variants. Overall, these experiments support the view that pausing alone is insufficient to mediate frameshifting and additional events are required. The phase dependence of pausing may be indicative of an activity in the ribosome that requires an optimal contact with mRNA secondary structures for efficient unwinding.

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