Expression of a coronavirus ribosomal frameshift signal in Escherichia coli: influence of tRNA anticodon modification on frameshifting.
Eukaryotic ribosomal frameshift signals generally contain two elements, a heptanucleotide slippery sequence (XXXYYYN) and an RNA secondary structure, often an RNA pseudoknot, located downstream. Frameshifting takes place at the slippery sequence by simultaneous slippage of two ribosome-bound tRNAs. All of the tRNAs that are predicted to decode frameshift sites in the ribosomal A-site (XXXYYYN) possess a hypermodified base in the anticodon-loop and it is conceivable that these modifications play a role in the frameshift process. To test this, we expressed slippery sequence variants of the coronavirus IBV frameshift signal in strains of Escherichia coli unable to modify fully either tRNA(Lys) or tRNA(Asn). At the slippery sequences UUUAAAC and UUUAAAU (underlined codon decoded by tRNA(Asn), anticodon 5' QUU 3'), frameshifting was very inefficient (2 to 3%) and in strains deficient in the biosynthesis of Q base, was increased (AAU) or decreased (AAC) only two-fold. In E. coli, therefore, hypomodification of tRNA(Asn) had little effect on frameshifting. The situation with the efficient slippery sequences UUUAAAA (15%) and UUUAAAG (40%) (underlined codon decoded by tRNA(Lys), anticodon 5' mnm5s2UUU 3') was more complex, since the wobble base of tRNA(Lys) is modified at two positions. Of four available mutants, only trmE (s2UUU) had a marked influence on frameshifting, increasing the efficiency of the process at the slippery sequence UUUAAAA. No effect on frameshifting was seen in trmC1 (cmnm5s2UUU) or trmC2 (nm5s2UUU) strains and only a very small reduction (at UUUAAAG) was observed in an asuE (mnm5UUU) strain. The slipperiness of tRNA(Lys), therefore, cannot be ascribed to a single modification site on the base. However, the data support a role for the amino group of the mnm5 substitution in shaping the anticodon structure. Whether these conclusions can be extended to eukaryotic translation systems is uncertain. Although E. coli ribosomes changed frame at the IBV signal (UUUAAAG) with an efficiency similar to that measured in reticulocyte lysates (40%), there were important qualitative differences. Frameshifting of prokaryotic ribosomes was pseudoknot-independent (although secondary structure dependent) and appeared to require slippage of only a single tRNA.
Brierley
I
I
Department of Pathology, University of Cambridge, UK.
Meredith
M R
MR
Bloys
A J
AJ
Hagervall
T G
TG
D000926:Anticodon / Q000235:genetics*; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics*; D018965:Frameshifting, Ribosomal / Q000235:genetics*; D001351:Infectious bronchitis virus / Q000235:genetics*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000235:genetics; D017354:Point Mutation; D012348:RNA, Transfer, Asn / Q000235:genetics; D012357:RNA, Transfer, Lys / Q000235:genetics; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics
D000926:Anticodon; D012348:RNA, Transfer, Asn; D012357:RNA, Transfer, Lys; D012367:RNA, Viral