ABSTRACT
The open reading frame SUP35 encoding the translation termination eRF3 factor vital to life contains three ATG codons (ATG1, ATG124, and ATG254). Previously, other authors detected two SUP35 transcripts: a major one that corresponds to the full-length open reading frame and a minor transcript that corresponds to the 3' terminal site of SUP35 starting at the third ATG codon (ATG254). In this work, mutations at triplets ATG1, ATG124, and ATG254 were obtained as well as double mutations, which combine the point mutation in one of three ATG triplets and a deletion at the site for binding with the transcription factor Abf1 within the SUP35 (sup35-deltaAbf1) promoter. The influence of these mutations on the yeast viability was analyzed. Mutations at triplets ATG124 and ATG254 did not affect yeast viability in their own right or in the background of deletion sup35-deltaAbf1. Mutation sup35-AGG1 (ATG1-->AGG) causes the lethal effect in cells grown on media containing glucose as the sole source of carbon. The replacement of glucose by galactose, or histidine starvation, partially restore the viability of sup35-AGG1 mutants, but not that of double mutants sup35-deltaAbf1,AGG1. The restoration of sup35-AGG1 mutant viability under these conditions can be explained by either the appearance (or enhancement) of the production of short peptides synthesized on the mRNA triplets SUP35 AUG124 and AUG254, or by the enhanced production of the full-length SUP35 transcript coupled with translation initiation from the noncanonical AGG1 codon. These data confirm that the expression of gene SUP35 at the transcription and(or) translation level is regulated by environmental conditions.
Subject(s)
Codon/metabolism , Mutation , Open Reading Frames/physiology , Peptide Chain Initiation, Translational/physiology , Prions/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Codon/genetics , Gene Expression Regulation, Fungal/physiology , Peptide Termination Factors , Prions/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/geneticsABSTRACT
Structural and functional organization of the 5' region of the SUP35 gene was analyzed in Saccharomyces cerevisiae yeast. Indirect DNA end labeling allowed two nuclease-hypersensitive sites and a region involved in nucleosomes to be revealed. DNase I and micrococcal nuclease hypersensitive sites were localized to almost the same regions: -461 ... -372 bp and -271 ... -91 bp for DNase I and -461 ... -356 bp and -231 ... -79 for micrococcal nuclease. Nucleosomes were localized to a region +22 ... +339 bp. Both the location of DNase I and micrococcal nuclease hypersensitive sites within the promoter region and the location of nucleosomes within the coding region remain the same at different cell culture growth phases. However, positioning nucleosomes were revealed within the SUP35 coding region only at the late logarithmic phase; the radioautographic pattern of them does not depend on the extent of nuclease digestion.
