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Triplex structures in an RNA pseudoknot enhance mechanical stability and increase efficiency of -1 ribosomal frameshifting.

ABSTRACT: Many viruses use programmed -1 ribosomal frameshifting to express defined ratios of structural and enzymatic proteins. Pseudoknot structures in messenger RNAs stimulate frameshifting in upstream slippery sequences. The detailed molecular determinants of pseudoknot mechanical stability and frameshifting efficiency are not well understood. Here we use single-molecule unfolding studies by optical tweezers, and frameshifting assays to elucidate how mechanical stability of a pseudoknot and its frameshifting efficiency are regulated by tertiary stem-loop interactions. Mechanical unfolding of a model pseudoknot and mutants designed to dissect specific interactions reveals that mechanical stability depends strongly on triplex structures formed by stem-loop interactions. Combining single-molecule and mutational studies facilitates the identification of pseudoknot folding intermediates. Average unfolding forces of the pseudoknot and mutants ranging from 50 to 22 picoNewtons correlated with frameshifting efficiencies ranging from 53% to 0%. Formation of major-groove and minor-groove triplex structures enhances pseudoknot stem stability and torsional resistance, and may thereby stimulate frameshifting. Better understanding of the molecular determinants of frameshifting efficiency may facilitate the development of anti-virus therapeutics targeting frameshifting.

SUBMITTER: Chen G

PROVIDER: S-EPMC2722267 | biostudies-literature | 2009 Aug

REPOSITORIES: biostudies-literature

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Triplex structures in an RNA pseudoknot enhance mechanical stability and increase efficiency of -1 ribosomal frameshifting.

Chen Gang G Chang Kung-Yao KY Chou Ming-Yuan MY Bustamante Carlos C Tinoco Ignacio I

Proceedings of the National Academy of Sciences of the United States of America 20090723 31

Many viruses use programmed -1 ribosomal frameshifting to express defined ratios of structural and enzymatic proteins. Pseudoknot structures in messenger RNAs stimulate frameshifting in upstream slippery sequences. The detailed molecular determinants of pseudoknot mechanical stability and frameshifting efficiency are not well understood. Here we use single-molecule unfolding studies by optical tweezers, and frameshifting assays to elucidate how mechanical stability of a pseudoknot and its frames ...[more]

PMID: 19628688

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Project description:RNA pseudoknots are structural elements that participate in a variety of biological processes. At -1 ribosomal frameshifting sites, several types of pseudoknot have been identified which differ in their organisation and functionality. The pseudoknot found in infectious bronchitis virus (IBV) is typical of those that possess a long stem 1 of 11-12 bp and a long loop 2 (30-164 nt). A second group of pseudoknots are distinguishable that contain stems of only 5 to 7 bp and shorter loops. The NMR structure of one such pseudoknot, that of mouse mammary tumor virus (MMTV), has revealed that it is kinked at the stem 1-stem 2 junction, and that this kinked conformation is essential for efficient frameshifting. We recently investigated the effect on frameshifting of modulating stem 1 length and stability in IBV-based pseudoknots, and found that a stem 1 with at least 11 bp was needed for efficient frameshifting. Here, we describe the sequence manipulations that are necessary to bypass the requirement for an 11 bp stem 1 and to convert a short non-functional IBV-derived pseudoknot into a highly efficient, kinked frameshifter pseudoknot. Simple insertion of an adenine residue at the stem 1-stem 2 junction (an essential feature of a kinked pseudoknot) was not sufficient to create a functional pseudoknot. An additional change was needed: efficient frameshifting was recovered only when the last nucleotide of loop 2 was changed from a G to an A. The requirement for an A at the end of loop 2 is consistent with a loop-helix contact similar to those described in other RNA tertiary structures. A mutational analysis of both partners of the proposed interaction, the loop 2 terminal adenine residue and two G.C pairs near the top of stem 1, revealed that the interaction was essential for efficient frameshifting. The specific requirement for a 3'-terminal A residue was lost when loop 2 was increased from 8 to 14 nt, suggesting that the loop-helix contact may be required only in those pseudoknots with a short loop 2.

Dataset Information

Triplex structures in an RNA pseudoknot enhance mechanical stability and increase efficiency of -1 ribosomal frameshifting.

Publications

Triplex structures in an RNA pseudoknot enhance mechanical stability and increase efficiency of -1 ribosomal frameshifting.

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