We have analyzed the expression behavior of endogenous genes in yeast using single-molecule analysis. For the first time, we have determined the exact number of mRNAs expressed in a single cell and used this information to model the expression kinetics for these genes. The key for these analyses was combining the number of cytoplasmic mRNAs present with the transcriptional status for each of the genes.
The ability to use cells without the need for any genetic modification is one main advantage of FISH. Cells are simply fixed, hybridized and analyzed. By this method, many cells can be analyzed and used for mathematical modeling. Additionally, placing FISH probes at different positions along the mRNA can be used to define the spacing between individual transcription-initiation events or to produce ‘footprints’ of polymerases on a gene (). Expanding this analysis by interrogating multiple genes simultaneously in the same cell will allow not only the dissection of single genes but also the study of co-regulatory networks and provide an important tool for systems analysis.
Our observation that mRNA abundance for most genes was higher than previously suggested was surprising, as these numbers were obtained by different hybridization techniques and are commonly used in the literature12,28,44,45
, although higher numbers have been suggested previously for a small subset of genes46
. The main reason for the discrepancy may lie in the normalization factor used by these studies, wherein it was assumed that a yeast cell expresses 15,000 mRNAs per cell. As shown in Supplementary Table 4 online
, the genes used in this study show a three- to six-fold higher expression than that determined previously12
. This would correct the number of transcripts to around 60,000 mRNAs per cell and indicates that the yeast transcriptome is more active than initially thought. This number also fits measurements suggesting that about 85% of the 200,000 yeast ribosomes are associated with mRNAs at an average ribosome density of 1 ribosome per 154 nt47,48
. Our observations also illustrate the utility of tools that enable the absolute quantification of gene expression, independently of ensemble measurements that use calibration and normalization factors.
Analyzing the expression of constitutively active genes revealed that mRNA variation is low, almost to a level that would be expected from pure Poisson noise. Although theoretical work has shown that different expression modes can lead to similar distributions30
, we show that expression is achieved by single, temporally well-separated initiation events, but not by transcription bursts. Even the cell cycle–regulated POL1
gene is expressed in a similar manner to a constitutive gene during its active period. With respect to promoter kinetics, this indicates that the assembly of an entire transcription complex usually leads to the initiation of a single transcript before the complex falls apart.
Recent work suggests that transcription complexes in general might not be as stable as thought. Even if transcription factors interact stably with their specific binding sites in vitro
, the residence time of many transcription factors at promoters in vivo
. However, for some classes of genes and promoters, factors that stabilize promoter complexes might allow the production of multiple mRNAs from a preassembled and stabilized complex. Transcription re-initiation has long been assumed to be required for efficient transcription from a promoter3,51,52
. Transcription bursts found for the PDR5
gene or for genes in higher eukaryotes might depend on factors allowing transcription re-initiation. Many genes in yeast showing high expression variation in protein levels are regulated by SAGA and contain a well-conserved TATA box, which is unusual for genes in yeast. Notably, it had previously been suggested that more stable binding of a TBP–TFIID complex, caused by the conserved TATA box, leads to re-initation–competent complexes, thereby causing transcriptional bursting6,9
. Consistent with this, mutating the TATA box in yeast has been shown to affect expression and reduce protein variation8,16,53
shows the parameter space for each gene tested, with the initiation rate c
normalized by the mRNA decay rate d
. Although some genes (MDN1
) have a less-restricted parameter space than others (POL1
), these genes all overlap in the nonbursting limit, whereas PDR5
is much less restricted. RNA polymerase II in mammalian cells and a bursting, artificial gene in bacteria are shown for comparison (the parameter space depicted for these two genes is only schematic). So, why has yeast but not higher eukaryotes chosen a constitutive expression mode for housekeeping genes? The possible explanation might lie in the fact that yeast is a rapidly dividing single cell. In higher eukaryotes, although mRNA variation is high owing to transcriptional bursting, the final protein variation is relatively low because mRNA noise is damped out by long mRNA and protein half-lives15
. In yeast, however, such buffering is not possible, as the average protein half-life is short and only twice as long as the average mRNA half-life54,55
. Maintaining constant expression is therefore better achieved by nonbursting, low-variation expression that constantly produces new proteins. Constant protein production is achieved by efficient translation, as most mRNAs (>70%) in a yeast cell are also polysome associated47
. However, in some cases, when fast responses are more important than precise control of transcriptional amplitudes, for example, during stress responses, bursting expression might be beneficial56
. Notably, bursting as well as constitutive RNA expression have been described in bacteria21,57
It is reasonable to speculate that the simple structure of yeast promoters, when compared to promoters in higher eukaryotes makes it easier to assemble transcription complexes for single initiation events. Promoters are often only a few hundred base pairs long and consist mainly of the histone-free region just upstream of the transcription start site58,59
. Opening promoters and assembling a transcription-competent complex is likely to require much more effort for the cell in higher eukaryotes, so it might be advantageous to transcribe multiple mRNAs once a complex is assembled, especially as higher total mRNA numbers are required as well60
. However, genes may exist in higher eukaryotes that are expressed in a less bursting and more constitutive manner. Future studies will show how other eukaryotes have evolved their modes of transcription and whether higher eukaryotes use transcription bursting only to express their transcriptome or if constitutive expression also exists. Single-molecule approaches such as that presented here will be essential to understand the kinetics of gene expression.