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Proc Natl Acad Sci U S A. May 28, 1996; 93(11): 5208–5212.
PMCID: PMC39223

Absolute mRNA levels and transcriptional initiation rates in Saccharomyces cerevisiae.

Abstract

We quantitate the absolute levels of individual mRNAs per yeast cell by hybridizing total yeast RNA with an excess of gene-specific 32P-oligonucleotides, and digesting the resulting RNA-DNA hybrids with S1 nuclease. By comparing the his3 hybridization signal from a known amount of yeast cells to the signal generated by a known amount of his3 RNA synthesized in vitro, we determine that yeast strain KY114 growing in yeast extract/peptone/glucose medium at 30 degrees C contains seven molecules of his3 mRNA per cell. Using a galactose shut-off procedure, we determined that the half-life of his3 mRNA is approximately 11 min under these conditions. From these observations, we calculate that one his3 mRNA molecule is synthesized every 140 s. Analysis of other his3 promoter derivatives suggests that the maximal transcriptional initiation rate in yeast cells is one mRNA molecule every 6-8 s. Using his3 as an internal standard, the number of mRNA molecules per cell have been determined for ded1, trp3, rps4, and gall under a variety of growth conditions. From these results, the absolute mRNA level of any yeast gene can be determined in a single hybridization experiment. Moreover, the rate of transcriptional initiation can be determined for mRNAs whose decay rates are known.

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  • St John TP, Davis RW. Isolation of galactose-inducible DNA sequences from Saccharomyces cerevisiae by differential plaque filter hybridization. Cell. 1979 Feb;16(2):443–452. [PubMed]
  • Struhl K, Davis RW. Transcription of the his3 gene region in Saccharomyces cerevisiae. J Mol Biol. 1981 Nov 5;152(3):535–552. [PubMed]
  • Bach ML, Lacroute F, Botstein D. Evidence for transcriptional regulation of orotidine-5'-phosphate decarboxylase in yeast by hybridization of mRNA to the yeast structural gene cloned in Escherichia coli. Proc Natl Acad Sci U S A. 1979 Jan;76(1):386–390. [PMC free article] [PubMed]
  • Kim CH, Warner JR. Messenger RNA for ribosomal proteins in yeast. J Mol Biol. 1983 Mar 25;165(1):79–89. [PubMed]
  • Herrick D, Parker R, Jacobson A. Identification and comparison of stable and unstable mRNAs in Saccharomyces cerevisiae. Mol Cell Biol. 1990 May;10(5):2269–2284. [PMC free article] [PubMed]
  • Parker R, Herrick D, Peltz SW, Jacobson A. Measurement of mRNA decay rates in Saccharomyces cerevisiae. Methods Enzymol. 1991;194:415–423. [PubMed]
  • Chen W, Tabor S, Struhl K. Distinguishing between mechanisms of eukaryotic transcriptional activation with bacteriophage T7 RNA polymerase. Cell. 1987 Sep 25;50(7):1047–1055. [PubMed]
  • Struhl K. Genetic properties and chromatin structure of the yeast gal regulatory element: an enhancer-like sequence. Proc Natl Acad Sci U S A. 1984 Dec;81(24):7865–7869. [PMC free article] [PubMed]
  • Chen W, Struhl K. Saturation mutagenesis of a yeast his3 "TATA element": genetic evidence for a specific TATA-binding protein. Proc Natl Acad Sci U S A. 1988 Apr;85(8):2691–2695. [PMC free article] [PubMed]
  • Struhl K. Nucleotide sequence and transcriptional mapping of the yeast pet56-his3-ded1 gene region. Nucleic Acids Res. 1985 Dec 9;13(23):8587–8601. [PMC free article] [PubMed]
  • Caponigro G, Muhlrad D, Parker R. A small segment of the MAT alpha 1 transcript promotes mRNA decay in Saccharomyces cerevisiae: a stimulatory role for rare codons. Mol Cell Biol. 1993 Sep;13(9):5141–5148. [PMC free article] [PubMed]
  • Iyer V, Struhl K. Mechanism of differential utilization of the his3 TR and TC TATA elements. Mol Cell Biol. 1995 Dec;15(12):7059–7066. [PMC free article] [PubMed]
  • Iyer V, Struhl K. Poly(dA:dT), a ubiquitous promoter element that stimulates transcription via its intrinsic DNA structure. EMBO J. 1995 Jun 1;14(11):2570–2579. [PMC free article] [PubMed]
  • O'Brien T, Lis JT. RNA polymerase II pauses at the 5' end of the transcriptionally induced Drosophila hsp70 gene. Mol Cell Biol. 1991 Oct;11(10):5285–5290. [PMC free article] [PubMed]
  • O'Brien T, Lis JT. Rapid changes in Drosophila transcription after an instantaneous heat shock. Mol Cell Biol. 1993 Jun;13(6):3456–3463. [PMC free article] [PubMed]
  • Kennell D, Riezman H. Transcription and translation initiation frequencies of the Escherichia coli lac operon. J Mol Biol. 1977 Jul;114(1):1–21. [PubMed]
  • Martin CT, Coleman JE. Kinetic analysis of T7 RNA polymerase-promoter interactions with small synthetic promoters. Biochemistry. 1987 May 19;26(10):2690–2696. [PubMed]

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