Gene expression is a central process to all lives. At the individual cell level, gene expression can often be viewed as a single molecule process, because many genes exist in one or a few copies per cell.

Taking the lac operon of E. coli for example, transcription by RNA polymerase is initiated upon a stochastic dissociation event of the repressor from the operator region of DNA, generating one mRNA molecule. A burst of fusion protein molecules will be synthesized by multiple ribosome molecules bound to the mRNA, yielding fluctuating protein production in time (Fig. 1A). In order to observe these processes in a cell, methods of protein detection with extremely highly sensitivity is required.

(A)	(B)

Fig.1 (A) Scheme of live-cell observations of gene expression. Transcription of one mRNA by an RNA polymerase results from an infrequent and transient dissociation event of repressor from DNA. Multiple copies of protein molecules are translated from the mRNA by ribosomes. Upon being assembled into E. coli's inner membrane, Tsr-Venus protein molecules can be detected individually by a fluorescence microscope. (B) A DIC/fluorescence overlay image of E. coli expressing Tsr-Venus from lac operon. Single Tsr-Venus molecules are shown in yellow dots.

 

We are working on two methodologies for probing gene expression in living cells with single protein molecule sensitivity.

In the first approach, we use a fast maturing fluorescent protein called Venus as a gene expression reporter. The key for achieving single molecule sensitivity was to immobilize the fluorescent protein reporter on the cell membrane, by constructing a chimeric fluorescent protein reporter tsr-venus, which contains a membrane localization sequence. It is normally difficult to detect single protein molecules inside cytoplasm - their fluorescence is spread by fast diffusion to the entire cell during the image acquisition time, and overwhelmed by the strong cellular autofluorescence. However molecules on cell membrane diffuse much slower, and therefore can be detected individually with our sensitive microscope. Using this approach, we recorded movies of growing E. coli cells to study the real-time expression from the lac operon in repressed state in real-time (Fig. 1B).

Another approach is based on the enzymatic reporter b -galactosidase. A single copy of b -galactosidase generates many copies of fluorescence molecules when a cell trapped in a microfluidic device was treated with a fluorogenic substrate, resulting enzymatic amplification in signal and single molecule sensitivity. This technique has been applied to probe gene expression from E. coli as well as individual budding yeast and mouse embryonic stem cells, demonstrating its generality.

Fig 2. (a) Schematic diagram of the two-layer microfluidic chamber used for the enzymatic assay. Cells are trapped inside a volume of 100 pl chamber. (b) Enzymatic reaction: hydrolysis of the synthetic substrate FDG by the b -gal yields a fluorescent product, fluorescein. (c) Real-time detection of b -gal production in a living cell. Discrete jumps in b -gal number are due to burst-like production of proteins. (d) Histogram of copy number of b -gal molecules per burst. The black line is the single exponential fit to the histogram

We monitor the expression of our reporters Tsr-YFP and the native b -galactosidase, respectively, from the repressed lac promoter on the chromosomal DNA. With both reporter systems, we observe that reporter protein production occurs in bursts, with the number of molecules per burst following an exponential distribution.  These observations were predicted only theoretical previously, accounted for by the competition between mRNA degradation by nuclease and translation by ribosome.  Our single-molecule experiment demonstrates the ability to provide quantitative real-time information on gene expression in a live cell.

We note that many important proteins are expressed at low levels, thus inaccessible by current genomic and proteomic techniques.  The single molecule sensitive reporter systems we developed open up possibilities for system wide characterization of the expression of these low copy number proteins.

 

 

Selected References:

Cai, Long; Friedman, Nir; Xie, X. Sunney; "Stochastic protein expression in individual cells at the single molecule level," Nature, 440, 358-362 (2006).

Yu, Ji; Xiao, Jie; Ren, Xiaojia; Lao, Kaiqin; Xie, X. Sunney; "Probing Gene Expression in Live Cells, One Protein Molecule at a Time," Science, 311, 1600-1603 (2006).