Absolute mRNA levels and transcriptional initiation rates in Saccharomyces cerevisiae
Absolute mRNA levels and transcriptional initiation rates in Saccharomyces cerevisiae
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DOI：
10.1073/pnas.93.11.5208
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发表时间：
1996-05-28
影响因子：
11.1
通讯作者：
Struhl, K
中科院分区：
文献类型：
--
作者：
Iyer, V;Struhl, K
我们通过将酵母总RNA与过量的基因特异性p -32寡核苷酸杂交，并用S1核酸酶消化产生的RNA DNA杂交体，来定量每个酵母细胞中单个mrna的绝对水平。通过比较已知数量酵母细胞的his3杂交信号与体外合成的已知数量his3 RNA产生的信号，我们确定在酵母提取物/蛋白胨/葡萄糖培养基中生长的酵母菌KY114每个细胞含有7个his3 mRNA。使用半乳糖关闭程序，我们确定在这些条件下his3mrna的半衰期约为11分钟。根据这些观察，我们计算出每140秒合成一个his3mrna分子。对其他his3启动子衍生物的分析表明，酵母细胞中最大的转录起始率为每6-8 s产生一个mRNA分子。以his3为内标，测定了不同生长条件下ded1、trp3、rps4和gal1在每个细胞中的mRNA分子数。从这些结果，任何酵母基因的绝对mRNA水平可以确定在一个单一的杂交实验。此外，对于已知衰变速率的mrna，转录起始速率可以确定。
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.