Stem-loop structures can effectively substitute for an RNA pseudoknot in-1 ribosomal frameshifting

Biophysical Structural ChemistrySupramolecular & Biomaterials Chemistr

Structural parameters affecting the kinetics of RNA hairpin formation

There is little experimental knowledge on the sequence dependent rate of hairpin formation in RNA. We have therefore designed RNA sequences that can fold into either of two mutually exclusive hairpins and have determined the ratio of folding of the two conformations, using structure probing. This folding ratio reflects their respective folding rates. Changing one of the two loop sequences from a purine- to a pyrimidine-rich loop did increase its folding rate, which corresponds well with similar observations in DNA hairpins. However, neither changing one of the loops from a regular non-GNRA tetra-loop into a stable GNRA tetra-loop, nor increasing the loop size from 4 to 6 nt did affect the folding rate. The folding kinetics of these RNAs have also been simulated with the program ‘Kinfold’. These simulations were in agreement with the experimental results if the additional stabilization energies for stable tetra-loops were not taken into account. Despite the high stability of the stable tetra-loops, they apparently do not affect folding kinetics of these RNA hairpins. These results show that it is possible to experimentally determine relative folding rates of hairpins and to use these data to improve the computer-assisted simulation of the folding kinetics of stem–loop structures

Functional analysis of the SRV-1 RNA frameshifting pseudoknot

Simian retrovirus type-1 uses programmed ribosomal frameshifting to control expression of the Gag-Pol polyprotein from overlapping gag and pol open-reading frames. The frameshifting signal consists of a heptanucleotide slippery sequence and a downstream-located 12-base pair pseudoknot. The solution structure of this pseudoknot, previously solved by NMR [Michiels,P.J., Versleijen,A.A., Verlaan,P.W., Pleij,C.W., Hilbers,C.W. and Heus,H.A. (2001) Solution structure of the pseudoknot of SRV-1 RNA, involved in ribosomal frameshifting. J. Mol. Biol., 310, 1109–1123] has a classical H-type fold and forms an extended triple helix by interactions between loop 2 and the minor groove of stem 1 involving base–base and base–sugar contacts. A mutational analysis was performed to test the functional importance of the triple helix for −1 frameshifting in vitro. Changing bases in L2 or base pairs in S1 involved in a base triple resulted in a 2- to 5-fold decrease in frameshifting efficiency. Alterations in the length of L2 had adverse effects on frameshifting. The in vitro effects were well reproduced in vivo, although the effect of enlarging L2 was more dramatic in vivo. The putative role of refolding kinetics of frameshifter pseudoknots is discussed. Overall, the data emphasize the role of the triple helix in −1 frameshifting

Three-dimensional models of the tRNA-like 3' termini of some plant viral RNAs.

Various plant viral RNAs possess a 3' terminus with tRNA-like properties. These viral RNAs are charged with an amino acid upon incubation with the cognate aminoacyl-tRNA synthetase and ATP. We have studied the structure of end-labelled 3'-terminal fragments of turnip yellow mosaic virus RNA and brome mosaic virus RNA 2 with chemical modifications of the adenosine and cytidine residues and with enzymatic digestions using RNase T1, nuclease S1 and the double-strand-specific ribonuclease from cobra venom. The data indicate that the 3' termini of these plant viral RNAs lack a cloverleaf structure as found in classical tRNA. The three-dimensional folding, however, reveals a striking resemblance with classical tRNA. The models proposed are supported by phylogenetic data. Apparently distinct three-dimensional solutions have evolved to meet the requirements for faithful recognition by tRNA-specific enzymes. The way in which the aminoacyl acceptor arms of these tRNA-like structures are constructed reveal novel features in RNA folding which may have a bearing on the secondary and tertiary structures of RNA in general. The dynamic behaviour of brome mosaic virus RNA 2 in solution presumably is illustrative of conformational transitions, which RNAs generally undergo on changing the ionic conditions

Differential response to frameshift signals in eukaryotic and prokaryotic translational systems.

The genomic RNA of beet western yellows virus (BWYV) contains a potential translational frameshift signal in the overlap region of open reading frames ORF2 and ORF3. The signal, composed of a heptanucleotide slippery sequence and a downstream pseudoknot, is similar in appearance to those identified in retroviral RNAs. We have examined whether the proposed BWYV signal functions in frameshifting in three translational systems, i.c. in vitro in a reticulocyte lysate or a wheat germ extract and in vivo in E. coli. The efficiency of the signal in the eukaryotic system is low but significant, as it responds strongly to changes in either the slip sequence or the pseudoknot. In contrast, in E. coli there is hardly any response to the same changes. Replacing the slip sequence to the typical prokaryotic signal AAAAAAG yields more than 5% frameshift in E. coli. In this organism the frameshifting is highly sensitive to changes in the slip sequence but only slightly to disruption of the pseudoknot. The eukaryotic assay systems are barely sensitive to changes in either AAAAAAG or in the pseudoknot structure in this construct. We conclude that eukaryotic frameshift signals are not recognized by prokaryotes. On the other hand the typical prokaryotic slip sequence AAAAAAG does not lead to significant frameshifting in the eukaryote. In contrast to recent reports on the closely related potato leafroll virus (PLRV) we show that the frameshifting in BWYV is pseudoknot-dependent