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Structure-Function Model for Kissing Loop Interactions That Initiate Dimerization of Ty1 RNA.

ABSTRACT: The genomic RNA of the retrotransposon Ty1 is packaged as a dimer into virus-like particles. The 5' terminus of Ty1 RNA harbors cis-acting sequences required for translation initiation, packaging and initiation of reverse transcription (TIPIRT). To identify RNA motifs involved in dimerization and packaging, a structural model of the TIPIRT domain in vitro was developed from single-nucleotide resolution RNA structural data. In general agreement with previous models, the first 326 nucleotides of Ty1 RNA form a pseudoknot with a 7-bp stem (S1), a 1-nucleotide interhelical loop and an 8-bp stem (S2) that delineate two long, structured loops. Nucleotide substitutions that disrupt either pseudoknot stem greatly reduced helper-Ty1-mediated retrotransposition of a mini-Ty1, but only mutations in S2 destabilized mini-Ty1 RNA in cis and helper-Ty1 RNA in trans. Nested in different loops of the pseudoknot are two hairpins with complementary 7-nucleotide motifs at their apices. Nucleotide substitutions in either motif also reduced retrotransposition and destabilized mini- and helper-Ty1 RNA. Compensatory mutations that restore base-pairing in the S2 stem or between the hairpins rescued retrotransposition and RNA stability in cis and trans. These data inform a model whereby a Ty1 RNA kissing complex with two intermolecular kissing-loop interactions initiates dimerization and packaging.

SUBMITTER: Gamache ER

PROVIDER: S-EPMC5454406 | biostudies-literature | 2017 Apr

REPOSITORIES: biostudies-literature

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Structure-Function Model for Kissing Loop Interactions That Initiate Dimerization of Ty1 RNA.

Gamache Eric R ER Doh Jung H JH Ritz Justin J Laederach Alain A Bellaousov Stanislav S Mathews David H DH Curcio M Joan MJ

Viruses 20170426 5

The genomic RNA of the retrotransposon Ty1 is packaged as a dimer into virus-like particles. The 5' terminus of Ty1 RNA harbors <i>cis</i>-acting sequences required for translation initiation, packaging and initiation of reverse transcription (TIPIRT). To identify RNA motifs involved in dimerization and packaging, a structural model of the TIPIRT domain in vitro was developed from single-nucleotide resolution RNA structural data. In general agreement with previous models, the first 326 nucleotid ...[more]

PMID: 28445416

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Project description:A kissing loop is a highly stable complex formed by loop-loop base-pairing between two RNA hairpins. This common structural motif is utilized in a wide variety of RNA-mediated processes, including antisense recognition, substrate recognition in riboswitches, and viral replication. Recent work has shown that the Tar-Tar(*) complex, an archetypal kissing loop, can form without Mg(2+), so long as high concentrations of alkali chloride salts are present. Interestingly, the stability of the complex is found to decrease with increasing cation size. In this work, we used molecular simulations to develop a molecular-level understanding of the origins of the observed counterion specificity. The ionic atmosphere of the Tar-Tar(*) complex was examined in the presence of 800 mm (where m denotes molality) NaCl, KCl, or CsCl. We used spatial free-energy density profiles to analyze differences in counterion accumulation at different spatial extents from the RNA molecule. We found that the lowest free-energy levels, which are situated in the vicinity of the loop-loop interface, can accommodate roughly two counterions, irrespective of counterion type. However, as we moved into higher free-energy levels, which are farther from the loop-loop interface, we observed increased differences in the numbers of accumulated counterions, with this number being largest for Na(+) and smallest for Cs(+). We analyzed the source of these differences and were able to attribute these to two distinct features: The extent of partial dehydration varies based on cation type; the smaller the cation, the greater the degree of dehydration. While smaller ions bind their first-hydration-shell water molecules more tightly than larger ions, they are also able to shed these water molecules for stronger electrostatic interactions with the RNA molecule. Secondly, we observed a distinct asymmetry in the numbers of accumulated cations around each hairpin in the Tar-Tar(*) complex. We were able to ascribe this asymmetry to the presence of a guanine tract in the Tar hairpin, which facilitates partial dehydration of the counterions. However, the smaller ions compensate for this asymmetry by forming a belt around the loop-loop interface in intermediate free-energy levels. As a result, the degree of asymmetry in counterion accumulation around individual hairpins shows an inverse correlation with the experimentally observed cation specificity for the stability of Tar-Tar(*) (i.e., the smaller the asymmetry, the greater the experimentally observed stability). This in turn provides a plausible explanation for why the smaller cations help stabilize the Tar-Tar(*) complex better than the larger cations. These findings suggest that the specific sequence and structural features of the Tar-Tar(*) complex may be the source of the experimentally observed cation specificity in Tar-Tar(*) stability. Our results lead to testable predictions for how changes in sequence might alter the observed counterion specificity in kissing loop stability.

Dataset Information

Structure-Function Model for Kissing Loop Interactions That Initiate Dimerization of Ty1 RNA.

Publications

Structure-Function Model for Kissing Loop Interactions That Initiate Dimerization of Ty1 RNA.

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