With the inclusion of mRNA distributions, one can achieve a more complete description of gene expression than is possible by considering only protein distributions. However, mRNA levels are not a direct measure of transcription per se.
The inference of transcriptional dynamics that comes from counting mRNA in fixed cells is limited by the half-life of mRNA. For an mRNA with a 30 min half-life, the steady state cytoplasmic mRNA level reflects almost an hour of mRNA expression. However, transcriptional responses are often fast and, depending on the length of a gene, require only a few minutes to produce mRNA 29
Higher temporal resolution observation of transcription kinetics can be obtained only by measuring transcription directly. Single cell methods for studying transcription rely on the ability to detect nascent mRNAs. Using the MS2 system, Chubb et al. studied the expression of the developmentally regulated dscA
gene in Dictyostelium
(). The study found that transcription occurred in irregularly-spaced bursts, with the length and amplitude of these bursts staying fairly constant30
. Transcription of the yeast CUP1
gene on the other hand was shown to be achieved in a different manner. Upon induction, mRNA production was constant over the time of activation31
. The constant transcription was rather surprising when compared to the binding behavior of the transcriptional activator Ace1p that regulated CUP1
transcription. Using fluorescence recovery after photobleaching (FRAP, described in this issue by Lidke & Wilson) the authors showed that Ace1p bound only transiently to the CUP1
promoter, with a residence time of less than two min suggesting that constant rebinding of Ace1p was required to ensure efficient transcription.
The low stability of promoter complexes in living cells (determined by FRAP, and reviewed in 32
) appears to be a common phenomenon, and might be one important factor that defines transcription kinetics. Many activators have very short dwell times at the transcription site, some for only a few seconds, suggesting that activators do not have to be stably bound to their promoter to allow transcription33–36
. Their affinity however might regulate transcription frequency. Binding of the HSP activator which regulates Drosophila heat shock genes becomes very tight upon heat shock37
. Heat shock genes are very efficiently transcribed, with new transcripts initiated about every four seconds at full activation29
. It is possible that tight binding of activators allows efficient transcription but simultaneously reduces the flexibility to fine tune the transcriptional response. In addition, the position of activator binding sites with respect to histones was shown to affect both transcription initiation and transcription rate38,39
. To further underscore the dynamic, probing nature of molecular interactions at the gene, Darzacq et al. showed that only about 1% of polymerase-gene interactions lead to a completion of an mRNA40
. Thus, there seem to be many different dynamic ways to modulate the transcriptional outcome, and a combination of methods will likely be required to dissect this process, probably gene by gene.
A relatively simple expression control seems to occur at constitutively expressed housekeeping genes in yeast. Zenklusen and colleagues used single molecule resolution FISH to determine the exact number of nascent mRNAs on constitutively expressed genes3
. This analysis showed that on short genes expressed at a low level, only a single nascent mRNA is detected at the gene. At a transcription elongation velocity of less than 1kb per minute, this suggested that initiation of individual mRNAs was separated by minutes. Taken together with the stability of promoter complexes described above, it seems likely that assembled transcription factor complexes often fall apart after initiation of a single mRNA. Combining polymerase occupancy data (determined from nascent mRNA at a transcription site) with the counting of mRNAs within the same cell further allowed modeling of the expression kinetics of these genes and showed that individual initiation events were uncorrelated with each other for most genes3
. This simple regulation might suggest the existence of a stochastic limiting step that controls the expression behavior. Such a step may constitute the binding of an activator, opening of chromatin, assembly or stability of a pre-initiation complex or the efficiency of a polymerase to enter elongation. Measuring transcriptional responses in real time with single mRNA resolution will be necessary to dissect these different possibilities.