Ribosomal frameshifting in plants: a novel signal directs the -1 frameshift in the synthesis of the putative viral replicase of potato leafroll luteovirus.
The 5.8 kb RNA genome of potato leafroll luteovirus (PLRV) contains two overlapping open reading frames, ORF2a and ORF2b, which are characterized by helicase and RNA polymerase motifs, respectively, and possibly represent the viral replicase. Within the overlap, ORF2b lacks an AUG translational start codon and is therefore presumably translated by -1 ribosomal frameshifting as a transframe protein with ORF2a. This hypothesis was studied by introducing the putative frameshift region into an internal position of the beta-glucuronidase (GUS) gene and testing for the occurrence of frameshifting in vivo by transient expression of GUS activity in potato protoplasts as well as in vitro by translation in the reticulocyte system. Both experimental approaches demonstrate that a -1 frameshift occurs at a frequency of approximately 1%. Site-directed mutagenesis identified the frameshift region and the involvement of the novel heptanucleotide motif UUUAAAU in conjunction with an adjacent stem-loop structure. Part of this stem-loop encodes a basic region in the ORF2b moiety of the transframe protein which was shown by binding experiments with PLRV RNA to represent a nucleic acid-binding domain. These data support a possible biological significance of the frameshift to occur at this position of the large overlap by including the putative RNA template-binding site of the PLRV replicase in the ORF2a/ORF2b transframe protein.
Prüfer
D
D
Max-Planck-Institut für Züchtungsforschung, Köln, FRG.
Tacke
E
E
Schmitz
J
J
Kull
B
B
Kaufmann
A
A
Rohde
W
W
D000595:Amino Acid Sequence; D001483:Base Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D016368:Frameshift Mutation; D015870:Gene Expression; D005966:Glucuronidase / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009690:Nucleic Acid Conformation; D009696:Nucleic Acids / Q000378:metabolism; D016366:Open Reading Frames; D010942:Plant Viruses / Q000201:enzymology*; D014176:Protein Biosynthesis; D012324:RNA-Dependent RNA Polymerase / Q000096:biosynthesis*; D011993:Recombinant Fusion Proteins / Q000235:genetics; D012270:Ribosomes; D011198:Solanum tuberosum / Q000382:microbiology; D014158:Transcription, Genetic
D009696:Nucleic Acids; D011993:Recombinant Fusion Proteins; D012324:RNA-Dependent RNA Polymerase; D005966:Glucuronidase