Absolute mRNA levels and transcriptional initiation rates in Saccharomyces cerevisiae

被引:150
作者
Iyer, V [1 ]
Struhl, K [1 ]
机构
[1] HARVARD UNIV, SCH MED, DEPT BIOL CHEM & MOLEC PHARMACOL, BOSTON, MA 02115 USA
关键词
D O I
10.1073/pnas.93.11.5208
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
We quantitate the absolute levels of individual mRNAs per yeast cell by hybridizing total yeast RNA with an excess of gene-specific P-32-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 gal1 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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页码:5208 / 5212
页数:5
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