The authors went on to measure the intensity of the fluorescence emitted by the nascent chains at the reporter gene locus in real time, which correlates with the number of RNA polymerases present on the gene downstream of the MBS cassette. In the CMVpr
cell line, the intensity fluctuated slightly over time around its mean value, reflecting small variations in the number of elongating polymerases present. The CCND1pr
cell line displayed strikingly different behavior. Periods of intense transcriptional activity alternated with periods where no fluorescence was detected. These bursts of activity occurred over periods ranging from minutes to hours - the average duration of the ON state was 200 minutes, while the duration of the OFF state averaged 22 minutes. This is a direct observation showing that promoters not only tune the global output of transcription (the average level) but also its kinetics: the gene associated with the CMV
promoter remains on at all times, with a high number of engaged polymerases. The CCND1
promoter, in contrast, alternates between ON and OFF states, with an overall lower number of engaged polymerases. These bursts are not necessarily incompatible with previous observations of constant cyclin D1 transcription levels over the cell cycle [7
]: as the pulses occur at time scales shorter than that of the cell-cycle phases, fluctuations might average out over time to generate a constant output.
Kinetic modeling suggests that the differences observed in the number of nascent RNA chains at the gene results from variation in the initiation frequency (one transcript initiated every 22 seconds for the CMVpr compared with one every 52 seconds for the CCND1pr), rather than to differences in elongation rate. These results confirm the common view that the main lever for transcription regulation is the efficiency of the promoter-dependent recruitment of elongating complexes (PolII combined with its necessary cofactors).
Taken together, these quantitative measurements draw a detailed picture of the regulation of mRNA production from two different promoters. The initiation rate ti
combined with the mean number of molecules of a given mRNA per cell (m
) can be used to estimate the RNA's half life td
), which is 42 minutes for RNA transcribed from the CMV promoter and 32 minutes for the same RNA transcribed from the CCND1
promoter, values close to the 30 minutes estimated for endogenous CCND1
]. Interestingly, the lifetime of these mRNA molecules is longer than the OFF state of the CCND1
promoter: in this case, transcription does not shut down long enough to significantly deplete the cell of its mRNAs. The outcome for the CCND1
promoter is a wide but single-peaked distribution of the number of mRNAs per cell, and shows that OFF states are not a stable feature leading to a phenotype of low mRNA copy number, but instead contribute dynamically to regulate the number of mRNA molecules within the cell.
Pulses of transcription have already been observed in reporter systems in bacteria and in endogenous genes in the slime mold Dictyostelium discoideum
and in cultured mammalian cells, whereas data on constitutively expressed genes in yeast suggest that these are transcribed via a constitutive, single-step initiation process [9
]. Even if not the rule, bursts of transcription seem to be a possible mode of transcription regulation in higher eukaryotes. One possible advantage is that generating bursts of transcripts provides the cell with more regulatory options: increasing the average level of expression can be achieved by increasing the burst frequency, the burst duration or the number of molecules per burst. Each of these options can be governed by different molecular interactions involving different cofactors.
What could be the mechanism behind such transcription pulses? The fact that this phenomenon was only observed at one of the two promoters studied rules out bursting as an intrinsic feature of eukaryotic gene expression - for example, to effect a large-scale transition to 'open' transcribable chromatin. Low-frequency binding of transcription factors could be one explanation. Detailed statistics of the times spent in the OFF/ON states in the case of the CCND1
promoter could provide further information on the number of hidden biochemical steps leading to gene activation or shutdown, and therefore help to build a better model describing the kinetics of transcription and its contribution to mRNA copy-number variability [10