The effects of alterations within the 3' untranslated region of the pyruvate kinase messenger RNA upon its stability and translation in Saccharomyces cerevisiae.
A 53 basepair deletion was constructed within the 3' untranslated region (3' UTR) of the yeast pyruvate kinase (PYK) gene borne upon a centromeric plasmid. Various modular assemblies of the pUC13 polylinker DNA (single unit = 44 bp) were used to replace the deleted region, and the effects of these modifications upon both transcript stability and translation ascertained in yeast. The use of a differential probing stratagem, based on the hybridisation of specific oligonucleotides to either pUC13 polylinker or unaltered PYK 3' UTR sequences, allowed for discrimination between mutant (plasmid borne) and wild-type (chromosomal) PYK transcripts. In no construct was there any significant alteration in mRNA stability, but translation of the PYK mRNA was severely curtailed by truncation of the 3' UTR or the presence of a strong hairpin-loop structure in the 3' UTR. A specific mutation in the N-terminal coding sequences, which created a premature termination codon in both a 3' 'tagged' PYK plasmid and a PYK/LacZ fusion gene, aborted the translation of a majority of their transcripts but left their chemical half-lives unaltered. This observation is at variance with some previously published data (Losson & Lacroute (1979) Proc Natl Acad Sci USA 76, 5134; Pelsey & Lacroute (1984) Curr Genet 8, 277), but is consistent with our own earlier observation that there is no obvious link between ribosome loading and mRNA stability in yeast (Santiago et al. (1986) Nucleic Acids Res 14, 8347). Possible reasons for this disparity are discussed.
Purvis
I J
IJ
Institute of Genetics, University of Glasgow, UK.
Bettany
A J
AJ
Loughlin
L
L
Brown
A J
AJ
D005656:Fungal Proteins / Q000096:biosynthesis / Q000235:genetics*; D006207:Half-Life; D011132:Polyribosomes / Q000378:metabolism; D014176:Protein Biosynthesis; D011770:Pyruvate Kinase / Q000096:biosynthesis / Q000235:genetics*; D012331:RNA, Fungal / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism
D005656:Fungal Proteins; D012331:RNA, Fungal; D012333:RNA, Messenger; D011994:Recombinant Proteins; D011770:Pyruvate Kinase