The mechanism of an exceptional case of reinitiation after translation of a long ORF reveals why such events do not generally occur in mammalian mRNA translation.
The subgenomic mRNA of feline caliciviruses is bicistronic with the two cistrons overlapping by four nucleotides, ..AUGA. The upstream cistron encodes a 75-kDa major capsid protein precursor (pre-VP1), and the downstream cistron a 10-kDa minor capsid protein. The kinetics of translation in reticulocyte lysates show that the downstream cistron is translated by a termination-reinitiation process, which is unusual in not requiring eIF4G or the eIF4F complex. Reinitiation requires the 3'-terminal 87 nucleotides (nt) of the pre-VP1 ORF, but no other viral sequences. The reinitiation site is selected by virtue of its proximity to this 87-nt element, and not its proximity to the pre-VP1 ORF stop codon, although this must be located not more than approximately 30 nt downstream from the restart codon. This 87-nt element was shown to bind 40S ribosomal subunits and initiation factor eIF3, and addition of supplementary eIF3 enhanced reinitiation efficiency. Mutants defective in reinitiation showed reduced affinity for eIF3 or defective 40S subunit binding (or both). These results suggest a mechanism in which some of the eIF3/40S complexes formed during disassembly of post-termination ribosomes bind to this 87-nt element in a position appropriate for reinitiation following acquisition of an eIF2/GTP/Met-tRNA i ternary complex.
Department of Biochemistry, University of Cambridge, Cambridge CB2 1GA, United Kingdom.
Tuija A A
TA
Pöyry
Ann
A
Kaminski
Emma J
EJ
Connell
Christopher S
CS
Fraser
Richard J
RJ
Jackson
D000818:Animals; D001483:Base Sequence; D017927:Calicivirus, Feline / Q000235:genetics* / Q000378:metabolism*; D002415:Cats; D018387:Codon, Initiator / Q000235:genetics; D018388:Codon, Terminator / Q000235:genetics; D005796:Genes / Q000235:genetics; D016679:Genome, Viral; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D016366:Open Reading Frames; D010442:Peptide Chain Initiation, Translational; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics*; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics*; D012689:Sequence Homology, Nucleic Acid; D015678:Viral Structural Proteins / Q000096:biosynthesis / Q000235:genetics
D018387:Codon, Initiator; D018388:Codon, Terminator; D012333:RNA, Messenger; D012367:RNA, Viral; D015678:Viral Structural Proteins