The Q-base of asparaginyl-tRNA is dispensable for efficient -1 ribosomal frameshifting in eukaryotes.
The frameshift signal of the avian coronavirus infectious bronchitis virus (IBV) contains two cis-acting signals essential for efficient frameshifting, a heptameric slippery sequence (UUUAAAC) and an RNA pseudoknot structure located downstream. The frameshift takes place at the slippery sequence with the two ribosome-bound tRNAs slipping back simultaneously by one nucleotide from the zero phase (U UUA AAC) to the -1 phase (UUU AAA). Asparaginyl-tRNA, which decodes the A-site codon AAC, has the modified base Q at the wobble position of the anticodon (5' QUU 3') and it has been speculated that Q may be required for frameshifting. To test this, we measured frameshifting in cos cells that had been passaged in growth medium containing calf serum or horse serum. Growth in horse serum, which contains no free queuine, eliminates Q from the cellular tRNA population upon repeated passage. Over ten cell passages, however, we found no significant difference in frameshift efficiency between the cell types, arguing against a role for Q in frameshifting. We confirmed that the cells cultured in horse serum were devoid of Q by purifying tRNAs and assessing their Q-content by tRNA transglycosylase assays and coupled HPLC-mass spectroscopy. Supplementation of the growth medium of cells grown either on horse serum or calf serum with free queuine had no effect on frameshifting either. These findings were recapitulated in an in vitro system using rabbit reticulocyte lysates that had been largely depleted of endogenous tRNAs and resupplemented with Q-free or Q-containing tRNA populations. Thus Q-base is not required for frameshifting at the IBV signal and some other explanation is required to account for the slipperiness of eukaryotic asparaginyl-tRNA.
Marczinke
B
B
Division of Virology Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QP, UK.
Hagervall
T
T
Brierley
I
I
D000818:Animals; D020029:Base Pairing; D001483:Base Sequence; D019556:COS Cells; D002851:Chromatography, High Pressure Liquid; D018965:Frameshifting, Ribosomal; D001351:Infectious bronchitis virus / Q000235:genetics*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012349:RNA, Transfer, Asp / Q000737:chemistry / Q000235:genetics*; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics*
D012349:RNA, Transfer, Asp; D012367:RNA, Viral