Messenger RNAs transcribed from yeast linear cytoplasmic plasmids possess unconventional 5’ and 3’ UTRs and suggest a novel mechanism of translation

Václav Vopálenský; Michal Sýkora; Tomáš Mašek; Martin Pospíšek

(C) The resulting plasmid, pRKL2-2, contains a version of K2ORF3 with mutations in the coding region of the MTase active site (white asterisks) and two genes, aminoglycoside 3'phosphotransferase (coding for G418 resistance) and K2ORF2, that are artificially controlled by the pGKL1 promoters K1UCR2 and K1UCR1, respectively. Shades of gray indicate the level of transcript capping, as in Fig. 1 . (E) Analysis of 5' ends of pRKL2-2 plasmids revealed that non-template adenosine addition and mRNA cap synthesis are directed by linear plasmid promoters. K2ORF2 controlled by the K1UCR1 promoter produces fewer capped transcripts with a higher level of non-template 5' polyadenylation, similar to wild-type K1ORF1 transcripts and in contrast with K2ORF2 transcripts controlled by the natural promoter. The K1UCR2 promoter caused a similar degree of mRNA capping and 5' non-template polyadenylation when controlling either its own gene (K1ORF2) or a heterologous bacterial gene coding for aminoglycoside 3'-phosphotransferase (G418 resistance). Thirteen to twenty-six independent cDNA clones were analyzed in each experiment.