Probing Gene Expression in Live Cells, One Protein Molecule at a Time

Abstract

We directly observed real-time production of single protein molecules in individual Escherichia coli cells. A fusion protein of a fast-maturing yellow fluorescent protein (YFP) and a membrane-targeting peptide was expressed under a repressed condition. The membrane-localized YFP can be detected with single-molecule sensitivity. We found that the protein molecules are produced in bursts, with each burst originating from a stochastically transcribed single messenger RNA molecule, and that protein copy numbers in the bursts follow a geometric distribution. The quantitative study of low-level gene expression demonstrates the potential of single-molecule experiments in elucidating the workings of fundamental biological processes in living cells.

Effects of multimerization on the temporal variability of protein complex abundance

“…Translation is modeled by Reaction 3, where k P is the stochastic rate of translation initiation and M is the number of available RNA molecules [27]. …”

Tuning cell differentiation patterns and single cell dynamics by regulating proteins’ functionalities in a toggle switch

“…Values of delays (in s) were extracted from measurements of the gene tsrvenus in E. coli [51]. τ 1 accounts for the promoter open complex formation (completion of transcription initiation).…”

“…It follows a normal distribution with a mean of 40 s and standard deviation of 4s according to measurements in a lac promoter [31,27], used to drive the expression of the tsr-venus. Since the average rate of transcriptional elongation in E. coli is ∼ 50 nt/s, τ 2 is, on average, 90 s (τ 2 = τ 1 + 2500/50), given 2500 nt is the gene length [51]. Note that the delay associated with the time needed to form the ribosome binding site R i is also τ 1 (and not τ 2 as for a complete RNA molecule), since the time to form R i is almost identical to the promoter clearance time given that we model gene expression in prokaryotes, where translation is initiated when the ribosome binding site is formed [44,52].…”

“…In translation, the time needed for R i clearance, τ 3 , is set to 2 s [13]. Since the average translation rate is 15 amino acids per second, τ 4 is computed as τ 4 = τ 3 + 2500/45 = 58 s. The post-translational protein assembly process was observed to take 420 ± 140 s in [51], which are set, in accordance, as the values for τ 5 and τ 5std .…”

“…For example, the time needed to form the promoter complex [31] affects the toggling frequency of genetic toggle switches (TS) [42]. The delayed Stochastic Simulation Algorithm (delayed SSA) [45] (a modified version of the original SSA [16]), able to match gene expression at the single RNA and protein level [52,51], allows modeling multiple time delayed reactions [44], and thus is used here to simulate the dynamics of the GRN of each cell.…”