Central dogma at the single-molecule level in living cells

Abstract

Gene expression originates from individual DNA molecules within living cells. Like many single-molecule processes, gene expression and regulation are stochastic, that is, sporadic in time. This leads to heterogeneity in the messenger-RNA and protein copy numbers in a population of cells with identical genomes. With advanced single-cell fluorescence microscopy, it is now possible to quantify transcriptomes and proteomes with single-molecule sensitivity. Dynamic processes such as transcription-factor binding, transcription and translation can be monitored in real time, providing quantitative descriptions of the central dogma of molecular biology and the demonstration that a stochastic single-molecule event can determine the phenotype of a cell.

Figure 1. Stochastic nature of single-molecule processes

a, Optical imaging of single protein molecules at room temperature. In his 1976 work, Hirschfeld demonstrated the detection of single protein molecules using a fluorescence microscope. A line scan of eight protein molecules was recorded. Adapted from ref. . b, Stochastic turnovers of a single enzyme molecule. The fluorescence signal of a cholesterol oxidase molecule exhibits stochastic switching between a fluorescent (reduced flavin) and nonfluorescent (oxidized flavin) state as enzymatic turnovers take place. The waiting time before each switching event is highly variable owing to a single rate-limiting step. Adapted from ref. . c, Single-molecule DNA sequencing. A single DNA polymerase is used to sequence DNA by incorporating fluorescently labelled nucleotides of four different colors. Although each incorporation happens stochastically with variable waiting times, the overall time for DNA replication, which is a sum of many sequential steps, is narrowly distributed. a.u., arbitrary units; adapted from ref. .

Figure 2. Central dogma at the single-molecule level

In a living bacterial cell, there is usually one copy of a particular gene, which is regulated by transcription factors (TFs), and transcribed into mRNA and translated into protein. A rate-limiting event, such as TF binding and unbinding on DNA, in this single molecule process results in stochasticity. The expression levels of mRNA (middle panel) and protein (bottom panel) show temporal fluctuations in a single cell lineage. This gives rise to variations of mRNA and protein copy numbers among a population of cells at a particular time (right panels).

Figure 3. Methods for imaging single molecules in live cells

Single-molecule fluorescence can be imaged using multiple laser illumination geometries that reduce the probe volume. a, In wide-field illumination, the entire cell is subject to laser exposure. For bacterial cells that have small volume, no further probe volume reduction is necessary. b, With total internal reflection, only the region within a few hundred nanometres from the coverslip is illuminated. This method is often used to image single membrane proteins, but cannot detect molecules deep in the cells. c, Two-photon excitation suppresses out-of-focus background, but suffers from slower time resolution owing to the need of point scanning d, Sheet illumination has reduced background, as well as increased time resolution because it does not require point scanning.

Figure 4. Real-time measurements of gene expression with single-molecule sensitivity

a, Detection by localization. The cellular autofluorescence makes it difficult to detect a freely diffusing fluorescent protein. However, a localized single molecule can be imaged above the autofluorescence background. b, Detection of single transcription factors in live cells. A lac repressor (LacI) labelled with YFP can be imaged when bound to its operator site on DNA. The localized fluorescence disappears after dissociation caused by the inducer IPTG. DIC, differential interference contrast microscopy; adapted from ref. . c, Target search by a transcription factor. When the inducer IPTG is removed (dilution), the lac repressor begins to search for its operator site. After rebinding to the target, localized fluorescence appears, allowing measurement of the search time. Adapted from ref. . d, Real time observation of protein synthesis at low expression levels. Individual YFP fusion protein molecules are visualized after being immobilized to the cell membrane, are synthesized in bursts after intrinsic noise. Adapted from ref. . e, Copy numbers of mRNA and protein of the same gene, measured in the same cell, show little correlation, which is mostly due to the differences in the lifetimes of mRNA and protein. The protein copy number distribution follows a gamma distribution. Adapted from ref..

Figure 5. Phenotype switching due to a single-molecule event

a, Bistability of the lac operon. The positive feedback by the normally repressed lac permease (LacY) results in bimodal distribution at intermediate inducer concentration with two distinct phenotypes, fluorescent or not. b, Threshold for positive feedback. The copy-number distribution of LacY in uninduced cells is the same as that without inducer, suggesting that the typical leaky expression of LacY is not sufficient to trigger the positive feedback. c, A large burst of expression originates from the stochastic event of the complete dissociation of a single transcription factor from DNA. A large expression burst of LacY (~300 molecules) is necessary to trigger the positive feedback. The switching of the phenotype is attributed to the complete dissociation of a single transcription factor, LacI. This experiment shows that a low probability single-molecule event can determine cell fate. Adapted from ref. .