THE ROLE OF UPSTREAM SEQUENCES IN THE TRANSCRIPTION OF CYH2, A YEAST RIBOSOMAL PROTEIN GENE (REGULATION)
SCHWINDINGER, WILLIAM FRANCIS
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In the yeast Saccharomyces cerevisiae more than seventy different genes are coordinately regulated in order to synthesize ribosomal proteins commensurate with the needs of the cell. Previous studies have shown that under normal growth conditions these genes are transcribed at the same rate. Furthermore, transcription of this entire set of genes can be altered in response to changes in growth conditions, including nitrogen starvation, nutritional upshift and mild temperature shock.;To test the hypothesis that specific sequences adjacent to the ribosomal protein genes are responsible for this coordinate regulation, the promoter of CYH2 was examined in detail. This gene codes for ribosomal protein L29. A mutation in the CYH2 gene causes resistance to cycloheximide, an inhibitor of protein synthesis. A series of deletions from the 5' end of the promoter and a second series of small internal deletions within the promoter region were constructed. These deletions were placed upstream of a resistant allele of CYH2, and introduced into a cycloheximide sensitive yeast strain on multicopy plasmids. The expression of CYH2 in these strains was analyzed by growth on cycloheximide containing plates. The RNA isolated from these strains was analyzed by northern blots and primer extension. These studies demonstrated that the region between 295 and 181 bp upstream of the initiation codon is required for efficient transcription. Sequences homologous to this region have been identified upstream of nineteen of the twenty-one yeast ribosomal protein genes which have been sequenced. These studies also demonstrated that a region approximately 100 bp upstream of the initiation codon and sequences at the transcription initiation site are required for proper initiation of transcription. The effect of a mild temperature shock on transcription in these deletions was investigated by shifting cultures which were growing at 23(DEGREES)C to 36(DEGREES)C and preparing RNA at various intervals between 10 and 60 min after the shift. The RNA isolated in these studies was analyzed by filter hybridization and by northern blots. None of the deletions had any effect on the regulation of transcription in response to a temperature shock, suggesting that the same sequences required for transcription are also involved in its regulation.
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