We all know about the central dogma of life whereby genetic information on DNA gets transcribed to messenger RNA (mRNA) that gets translated into a protein. Based on this, in many studies it is assumed that measuring the mRNA levels in cells gives an indication about the amount of proteins. A recent work, published in Science, lead by the Xie group at Harvard demonstrates that in bacteria, while ensemble level mRNA numbers and their corresponding amounts of proteins correlate well, when these are measured at the level of a single cell there is zero correlation.
The authors explain that this is possibly because mRNAs have a short lifetime (minutes) while proteins often survive for hours and in-fact beyond the cell-division cycles.
Addtionally, the authors also demonstrate that there is a large cell to cell variation in gene-expression levels, the so-called ‘noise’, inherent across the whole genome.
While the results seem somewhat intuitive, it is difficult to prove experimentally. Monitoring single protein and mRNA levels in cells is not trivial, more so in bacterial cells due to the very low copy numbers. Hence an extremely sensitive technique is required. A few years back, the Xie lab had pioneered – among a plethora of innovative optical techniques – the ability to detect single molecule fluorescence in bacteria. By tagging a bacterial gene with a fluorescent protein and employing a powerful fluorescence microscope, they could practically observe each gene expression event by the flash of light from expressed fluorescent proteins (as shown below). In earlier studies they used this platform to investigate the stochastic manner in which genes are switched on.
In this particular study, the group used a bacterial library developed at University of Toronto to tag every gene of E. Coli with a fluorescent protein (the Yellow Fluorescent Protein in particular), obtaining a 25% coverage of the genome with 1018 genes tagged. By measuring the fluorescence from these tagged proteins, they could quantitatively determine the number of proteins per cell. Simultaneously, they measured the mRNA levels by introducing a fluorescently labeled oligonucleotide that hybridizes with the YFP mRNA [a technique called Fluorescence in-situ Hybridization (FISH)]. The use of a YFP-specific nuclei acid is a clever trick that keeps things simple by having to design only one probe for all the different mRNAs. To follow the expression levels of many genes at the same time, they employed a mirofluidic chamber that introduced bacterial strains, each with a different gene tagged, into 96 wells, where they were imaged automatically at a high-speed. Protein and mRNA levels were deduced by analyzing these images.
I have to admit that personally this paper is really much more exciting from the technical point of view. While visualization and quantification of single molecule fluorescence has been around since the late 90s, the combination of high-throughput molecular biology and imaging with the help of solid-state engineering (the microfluidics chamber) is quite a feat. Also, this is possibly the first system to carry out proteomic analysis and correlate with the transcritome at single cell levels (in bacteria at least).
From the standpoint of genetics, the significance lies in this being a springboard for further investigation of how expression noise affects genetic pathways, as noted by Sanjay Tyagi of New Jersey Medical School in an accompanying Perspective article:
Now that there is reliable knowledge of the levels of expression and the underlying variation of mRNAs and proteins for a considerable portion of the E. coli genome, it is possible to explore how noise propagates along gene expression pathways, in which the amount of one protein can influence the expression of another. Investigators can also investigate how cells coordinate the expression of proteins that need to work together, such as multisubunit proteins or proteins that serve within metabolic cycles.
1. The Xie lab maintains an excellent website that nicely describe their research. They also have pdf copies of all their papers for download. Not sure how kosher that is from copyright perspectives, but I am not complaining.