Plant luteoviral RNA viruses employ -1 frameshifting for the production of P1
and P1-P2 fusion proteins important for viral replication. Luteoviral pseudoknots are
characterized by three adenosines in the 3' side of loop L2 known to be important for
maintaining frameshifting efficiency and pseudoknot stability. A proposed P1-P2
mRNA pseudoknot from sugarcane yellow leaf virus (ScYLV) was of interest since it
contained two adenosine to cytidine substitutions in L2. Functional analysis shows that
the in vitro frameshifting efficiency is greater (~15%) than any other luteoviral
pseudoknot. The NMR-derived solution structure of the ScYLV RNA pseudoknot
shows that C25 is looped out of the triplex structure and the 3' most L2 cytidine (C27)
and A24 form cis Watson-Crick/sugar-edge interactions with C14 and C15 in stem S1,
respectively. Thus, the ScYLV pseudoknot maintains a similar triple helical architecture
as other luteoviral pseudoknots. Surprisingly, the frameshifting efficiency of the C27A
ScYLV pseudoknot is decreased by ~8 fold relative to wild-type ScYLV. The solution
structure of the C27A ScYLV RNA exhibits a global fold similar to the wild-type RNA;
however, distinct hydrogen bonding interactions at the helical junction are observed.
Specifically, C8+ in the C8+ major groove base triple moves ~2.3
relative to the accepting (G12-C28) base pair relative to the WT RNA. New NMR experiments
have been developed and/or applied to confirm Watson-Crick base pairs and tertiary
structural interactions in the PEMV-1 and ScYLV pseudoknots by direct observation of
trans hydrogen bond scalar couplings. In addition, intrabase couplings in cytidine and
adenosine have been measured, providing a valuable tool for the assignment of amino
and N3/N1 resonances in RNA. Finally, thermodynamic analysis of the pairwise
coupling between the major groove and minor groove tertiary structural hydrogen bonds
at the helical junction have been investigated by monitoring the thermal unfolding of
WT, dC14, C27A, and dC14/C27A RNAs as a function of pH. Favorable pairwise
coupling characterized the WT ScYLV and BWYV RNAs, while unfavorable coupling
characterized the poorly functional C27A ScYLV RNA. The implications of these
structural, functional, and thermodynamic findings on the mechanism of frameshift
stimulation is discussed.
| Identifer | oai:union.ndltd.org:tamu.edu/oai:repository.tamu.edu:1969.1/3328 |
| Date | 12 April 2006 |
| Creators | Cornish, Peter Verle |
| Contributors | Giedroc, David P. |
| Publisher | Texas A&M University |
| Source Sets | Texas A and M University |
| Language | en_US |
| Detected Language | English |
| Type | Book, Thesis, Electronic Dissertation, text |
| Format | 5874215 bytes, electronic, application/pdf, born digital |
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