Mutational analysis of the pseudoknot in the tRNA-like structure of turnip yellow mosaic virus RNA

Mans, R. M. W.; Steeg, M. H. Van; Verlaan, P. W. G.; Pleij, C.W.A.; Bosch, L.

doi:10.1016/0022-2836(92)90727-2

Cited by 25 publications

(47 citation statements)

References 37 publications

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“…Although there is no structural evidence for whether the U30-A13 base pair forms, it is more likely that this relatively weak base pair (compared with a G-C pair) does not form, thus leaving an unpaired A as in VPK. The only structural studies of pseudoknots are from direct biochemical probing of the pseudoknot of turnip yellow mosaic virus RNA (Mans et al, 1992), and from biochemical and NMR studies of synthetic RNA oligonucleotides of pseudoknots Wyatt et al, 1990). Consistent with this prediction, deleting the A in the junction of FIV ( Figure 7A, FIVAA13) and SRV ( Figure 7A, SRVAA13) severely reduced the frameshifting levels to 2 and 4%, respectively ( Figure 7B).…”

Section: Similar Pseudoknot Junctions In Other Retrovirusesmentioning

confidence: 67%

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.

et al. 1995

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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.

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Section: Similar Pseudoknot Junctions In Other Retrovirusesmentioning

confidence: 67%

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.

et al. 1995

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“…However, it was striking that a pseudoknot comprising exactly the same number and type of base pairs induced a greater degree of pausing. No theoretical framework exists as yet for calculation of the precise thermodynamic stability of a pseudoknot, but it seems likely that it is considerably less stable than a stem-loop with an equivalent number of base pairs (27,42), particularly if these base pairs are contiguous (38). Thus, the extent of pausing induced by the pseudoknot is unlikely to reflect simply the energy required for its unwinding but rather its peculiar architecture.…”

Section: Resultsmentioning

confidence: 99%

Ribosomal pausing during translation of an RNA pseudoknot.

et al. 1993

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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 pseudoknotforming 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.

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“…Pseudoknots are often thermodynamically metastable, existing in an equilibrium between the pseudoknot and a single stem-loop (Wyatt et al 1990;Mans et al 1992). When the pseudoknot form is present, the region downstream of the template might be held in a position that facilitates binding of primer and initiation of its elongation.…”

Section: Secondary Structure and Implications For Functionmentioning

confidence: 99%

Telomerase RNAs of different ciliates have a common secondary structure and a permuted template.

Lingner¹,

Hendrick²,

Cech³

1994

Genes Dev.

183

220

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Telomerase is composed of protein and RNA. The RNA serves as a template for telomere DNA synthesis and may also be important for enzyme structure or catalysis. We have used the presence of conserved sequence elements in the promoter and template regions to amplify by PCR the telomerase RNA genes from six different hypotrichous ciliates: Oxytricha nova, Oxytricha trifallax, Stylonychia mytilis, Stylonychia lemnae, Enplotes aedicalatns, and Euplotes eurystomns. RNaseH cleavage of the O. nova RNA in extracts by use of a complementary oligonucleotide leads to loss of telomerase activity, supporting the identification. Primary sequence and biochemical experiments suggest that the templates of Oxytricha and Stylonychia are circularly permuted relative to that of E. aediculatns. On the basis of the pause sites, the former two add G4T4 during a single primer elongation cycle, whereas E. aedicalatus adds G3T4G. The only primary sequence element outside the template that is conserved between these phylogenetically distant telomerase RNAs is the sequence 5'-(C)UGUCA-3', which precedes the template regions by exactly two bases. We propose a common secondary structure model that is based on nucleotide covariations, a model which resembles that proposed previously for tetrahymenine telomerase RNAs.

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Mutational analysis of the pseudoknot in the tRNA-like structure of turnip yellow mosaic virus RNA

Cited by 25 publications

References 37 publications

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.

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.

Ribosomal pausing during translation of an RNA pseudoknot.

Telomerase RNAs of different ciliates have a common secondary structure and a permuted template.

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