Initiation of translation at a UAG stop codon in the aldolase gene of Plasmodium falciparum.

Abstract

The gene coding for the key glycolytic enzyme fructose‐1,6‐diphosphate aldolase of the human malaria parasite Plasmodium falciparum lacks a functional AUG initiation codon for translation. Protein sequences of natural or in vitro translated aldolase include the candidate start methionine residue at internal positions. No additional AUG start codon is found in genomic DNA, cDNA or mRNA sequences. Instead, a UAG chain termination codon is recognized as the start signal of protein synthesis in vivo and in vitro.

In vitro translation of Plasmodium falciparum aldolase is not initiated at an unusual site.

“…They include the entire protein coding region and 160 bp downstream from the termination codon (Knapp et al, 1990). One clone (pAldTAG) contains identical nucleotides flanking the putative start codon to those in the aldolase cDNA clone described by Ghersa et al (1990). In another clone (pAldAAG) the UAG codon is converted to AAG.…”

“…Constructs pAIdAAG, pAldTAG and pAIdATG* encoded proteins of identical size ( Figure 2, lanes 1-3), whereas the product of pAIdATG was slightly larger (Figure 2, lane 4). The report by Ghersa et al (1990) is the first to describe initiation of translation at a UAG codon, a surprising finding since cell-free systems lack aminoacyl-tRNA molecules complementary to stop codons. This polypeptide migrated to the same position in each lane.…”

“…The most 5' in-frame AUG codon (AUG*) encoded by the genomic fragment isolated initially (Certa et al, 1988) does not represent the initiation of translation; sequencing of a protease V8 fragment of parasite aldolase isolated from infected erythrocytes revealed an additional six amino acids encoded by triplets immediately upstream of AUG* (Ghersa et al, 1990). Two models have been proposed to explain the biogenesis of parasite aldolase: Ghersa et al (1990) isolated four independent cDNA clones containing 5' sequences which encoded an in-frame UAG codon seven triplets upstream from AUG* but no other in-frame AUG. The authors confirmed this result by sequencing of the mRNA.…”

“…Two models have been proposed to explain the biogenesis of parasite aldolase: Ghersa et al (1990) isolated four independent cDNA clones containing 5' sequences which encoded an in-frame UAG codon seven triplets upstream from AUG* but no other in-frame AUG. Therefore the authors concluded that the UAG stop codon seven triplets upstream from AUG* was used as a translational start codon in vivo and in vitro (Ghersa et al, 1990). It appeared that expression of a cDNA clone in vitro resulted in a polypeptide of identical size to that of aldolase isolated from parasitized erythrocytes.…”

“…In order to investigate the molecular mechanisms which might lead to translational initiation at a termination codon, we reconstructed the cDNA clone used by Ghersa et al (1990) and introduced mutations at the site of the putative translational start codon. The DNA constructs were transcribed in vitro and the corresponding mRNAs were translated in a rabbit reticulocyte lysate.…”