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A loop 2 cytidine-stem 1 minor groove interaction as a positive determinant for pseudoknot-stimulated -1 ribosomal frameshifting.

ABSTRACT: The molecular determinants of stimulation of -1 programmed ribosomal frameshifting (-1 PRF) by RNA pseudoknots are poorly understood. Sugarcane yellow leaf virus (ScYLV) encodes a 28-nt mRNA pseudoknot that promotes -1 PRF between the P1 (protease) and P2 (polymerase) genes in plant luteoviruses. The solution structure of the ScYLV pseudoknot reveals a well ordered loop 2 (L2) that exhibits continuous stacking of A20 through C27 in the minor groove of the upper stem 1 (S1), with C25 flipped out of the triple-stranded stack. Five consecutive triple base pairs flank the helical junction where the 3' nucleotide of L2, C27, adopts a cytidine 27 N3-cytidine 14 2'-OH hydrogen bonding interaction with the C14-G7 base pair. This interaction is isosteric with the adenosine N1-2'-OH interaction in the related mRNA from beet western yellows virus (BWYV); however, the ScYLV and BWYV mRNA structures differ in their detailed L2-S1 hydrogen bonding and L2 stacking interactions. Functional analyses of ScYLV/BWYV chimeric pseudoknots reveal that the ScYLV RNA stimulates a higher level of -1 PRF (15 +/- 2%) relative to the BWYV pseudoknot (6 +/- 1%), a difference traced largely to the identity of the 3' nucleotide of L2 (C27 vs. A25 in BWYV). Strikingly, C27A ScYLV RNA is a poor frameshift stimulator (2.0%) and is destabilized by approximately 1.5 kcal x mol(-1) (pH 7.0, 37 degrees C) with respect to the wild-type pseudoknot. These studies establish that the precise network of weak interactions nearest the helical junction in structurally similar pseudoknots make an important contribution to setting the frameshift efficiency in mRNAs.

SUBMITTER: Cornish PV

PROVIDER: S-EPMC1200304 | biostudies-literature | 2005 Sep

REPOSITORIES: biostudies-literature

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Publications

A loop 2 cytidine-stem 1 minor groove interaction as a positive determinant for pseudoknot-stimulated -1 ribosomal frameshifting.

Cornish Peter V PV Hennig Mirko M Giedroc David P DP

Proceedings of the National Academy of Sciences of the United States of America 20050825 36

The molecular determinants of stimulation of -1 programmed ribosomal frameshifting (-1 PRF) by RNA pseudoknots are poorly understood. Sugarcane yellow leaf virus (ScYLV) encodes a 28-nt mRNA pseudoknot that promotes -1 PRF between the P1 (protease) and P2 (polymerase) genes in plant luteoviruses. The solution structure of the ScYLV pseudoknot reveals a well ordered loop 2 (L2) that exhibits continuous stacking of A20 through C27 in the minor groove of the upper stem 1 (S1), with C25 flipped out ...[more]

PMID: 16123125

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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

A loop 2 cytidine-stem 1 minor groove interaction as a positive determinant for pseudoknot-stimulated -1 ribosomal frameshifting.

Publications

A loop 2 cytidine-stem 1 minor groove interaction as a positive determinant for pseudoknot-stimulated -1 ribosomal frameshifting.

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