Modulation of translational efficiency by contextual nucleotides flanking a baculovirus initiator AUG codon.
In a previous study of translational regulation of a baculovirus gene, we observed that translation initiated at an unexpectedly high efficiency from an AUG codon found in what was believed to be a poor context (M.-J. Chang and G. W. Blissard, 1997, J. Virol. 71, 7448-7460). In the current study, we examined the roles of nucleotides flanking a baculovirus AUG initiator codon in modulating translation initiation in lepidopteran insect cells. The roles of nucleotides flanking the AcMNPV gp64 initiator codon were examined by site-directed mutagenesis and functional assays in transfected Sf9 cells. To eliminate potential cis-acting sequences and effects, the gp64 initiator context was cloned in-frame with a chloramphenicol acetyl transferase reporter gene and under the control of a heterologous promoter. All possible single-nucleotide substitutions were generated in positions -6 to -1 and +4 to +6, relative to the A of the initiator AUG codon, which was designated +1. Constructs were transfected into lepidopteran cells and translation products were quantified by an enzyme-linked immunosorbent assay procedure. Substitutions of pyrimidines or other nucleotides at the -3 position resulted in little or no detectable effect on translation efficiency. In contrast, specific substitutions at the +4 and +5 positions resulted in approximately 2- to 3-fold increases in translation. Substitution of A in the +4 position resulted in an approximately 3-fold increase in translation, and substitution of any nucleotide for T in the +5 position resulted in approximately 1.9- to 2.8-fold increases. Substitutions at other positions (-6 to -1 and +6) resulted in no detectable increase or decrease in translation efficiency. These experimental results suggest an optimal initiator context of 5'-N N N N N N A U G A a/c/g N-3' for efficient translation initiation in lepidopteran cells. Consensus translation initiation contexts were generated from baculovirus genes and lepidopteran genes, then compared with the experimental results from the gp64 initiator context.
Chang
M J
MJ
Boyce Thompson Institute at Cornell University, Tower Road, Ithaca, New York, 14853-1801, USA.
Kuzio
J
J
Blissard
G W
GW
D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D018387:Codon, Initiator / Q000235:genetics*; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D007915:Lepidoptera / Q000235:genetics*; D008969:Molecular Sequence Data; D017924:Nucleopolyhedroviruses / Q000235:genetics*; D009711:Nucleotides / Q000235:genetics*; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D014158:Transcription, Genetic; D014162:Transfection
D018387:Codon, Initiator; D009711:Nucleotides