Figure 3.

Small and large bursts in the absence of positive feedback. In order to eliminate positive feedback from permease transport, we constructed strain SX701, replacing the lactose permease with the membrane protein fusion, Tsr-YFP. (A) Real-time traces of protein production in SX701 in 200 µM TMG. The FPs are photobleached immediately after detection to ensure that only newly produced proteins are measured after each 4 minute interval. Representative traces of single cells show frequent small bursts, associated with partial dissociation events, and a rare large burst, associated with a complete dissociation event, of protein production. (B) Distribution of burst sizes determined for 208 bursts from the real-time experiment depicted in B. Although the majority of bursts are small, a number of unusually large bursts are observed. The former are attributed to partial dissociations, and the latter to complete dissociations of the tetrameric repressor. Inset: The occurrence of burst sizes smaller than 10 molecules is well-fit by an exponential distribution (red line). (C) Analysis of small bursts from partial dissociations of the tetrameric repressor shows that the distribution of YFP molecules in the range of zero to ten molecules does not change in the range of 0 to 200 µM TMG. A small percentage of cells have much more than ten molecules and do not appear on the axis of this plot (see Fig. S7C). Over 100 cells were analyzed for each concentration. Inset: The frequency of bursts per cell cycle, (purple circles) increases slightly while the average number of proteins per burst, (gray triangles), remains approximately constant. These kinetic parameters are determined from the steady-state distribution of YFP in those cells with less than ten molecules (see text). Error bars are standard errors determined by bootstrapping. Gamma distributions using the determined parameters are overlayed as dashed lines. The gamma distribution for 200 µM is normalized for the subpopulation of cells with less than 10 molecules. (D) In addition to having the permease replaced with Tsr-YFP, strain SX703 has the auxiliary operators removed, eliminating DNA looping so that every dissociation event is a complete dissociation. The protein number distributions should reflect the bursts of protein expression from complete dissociations alone. Inset: the noise parameters µ²/σ² (purple circles ) and σ²/µ (gray triangles) suggest that the frequency of large bursts is independent of the TMG concentration but that the burst size is not.