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.
Garcia
A
A
Department of Biochemistry, Gorlaeus Laboratories, Leiden University, The Netherlands.
van Duin
J
J
Pleij
C W
CW
D001483:Base Sequence; D004247:DNA; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D005786:Gene Expression Regulation; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010942:Plant Viruses / Q000235:genetics*; D014176:Protein Biosynthesis; D012313:RNA; D014908:Triticum / Q000235:genetics*
D012313:RNA; D004247:DNA