Quantifying E. coli Proteome and Transcriptome with Single-Molecule Sensitivity in Single Cells

Abstract

Protein and mRNA copy numbers vary from cell to cell in isogenic bacterial populations. However, these molecules often exist in low copy numbers, and are difficult to detect in single cells. Here we carried out quantitative system-wide analyses of protein and mRNA expression in individual cells with single-molecule sensitivity using a newly constructed yellow fluorescent protein fusion library for Escherichia coli. We found that almost all protein number distributions can be described by the gamma distribution with two fitting parameters which, at low expression levels, have clear physical interpretations as the transcription rate and protein burst size. At high expression levels, the distributions are dominated by extrinsic noise. Strikingly, we found that a single cell's protein and mRNA copy numbers for any given gene are uncorrelated.Gene expression is often stochastic because gene regulation takes place at a single DNA locus within a cell. Such stochasticity is manifested in fluctuations of mRNA and protein copy numbers within a cell lineage over time, and in variations of mRNA and protein copy numbers among a population of genetically identical cells at a particular time (1,2,3,4). Because both manifestations of stochasticity are connected, measurement of the latter allows the deduction of the gene expression dynamics in a cell (5). We aim to characterize such mRNA and protein distributions in single bacteria cells at a system-wide level.While single cell mRNA profiling has been carried out with cDNA microarray (6) and mRNA-seq (7), these studies did not have single molecule sensitivity and are not suitable for bacteria, which express mRNA at low copy numbers (8). A fluorescent protein reporter library of Saccharomyces cerevisiae (9) has proven to be extremely useful in protein profiling (10,11). However, the lack of sensitivity in existing flow cytometry or fluorescence microscopy techniques prevented the quantification of one third of the labeled proteins because of their low copy numbers. In recent years, single-molecule fluorescence ** Publisher's Disclaimer: This manuscript has been accepted for publication in Science. This version has not undergone final editing. Please refer to the complete version of record at http://www.sciencemag.org/. The manuscript may not be reproduced or used in any manner that does not fall within the fair use provisions of the Copyright Act without the prior, written permission of AAAS." † To whom correspondence should be addressed. xie@chemistry.harvard.edu. * These authors contributed equally to this work. NIH Public Access Author ManuscriptScience. Author manuscript; available in PMC 2010 August 17. Single-molecule imaging of a YFP reporter libraryWe created a chromosomal YFP fusion library (Fig. 1A), in which each strain has a particular gene tagged with the YFP coding sequence. YFP can be detected with single molecule sensitivity in live bacterial cells (8,18). We converted the C-terminus tags of an existing chromosomally affinity-tagged E. coli library (19,20...

Effects of multimerization on the temporal variability of protein complex abundance

“…Setting α M = 1 yields Poisson distributed mRNA numbers M ~ Poi( k M d M -1 ), while α M > 1 and α M < 1 result in sub- and super-Poissonian distributions of RNA numbers, respectively (both of which have been reported in E. coli [6,20]). We note that, for integer values of α M , the parameter has a physical interpretation: namely, it represents a sequential process with α M elementary steps, each of duration ( α M k M ) -1 , which is in accordance with a sequential process of transcription initiation [1].…”

“…the open complex formation that involves structural changes of the DNA). Similarly, super-Poissonian RNA dynamics [6] ( α M < 1) require the existence of some additional mechanisms, such as a two-state model of transcription [26]. …”

“…Finally, the degradation of proteins is modeled via Reaction 4, a first order process [6]. (The rate of protein degradation has been observed to be constant, and identical in different growth conditions [29].…”

“…There are several mechanisms responsible for regulating their numbers in cells, which act at various stages of protein production [1-4], activation [5], and degradation. A recent study has provided genome-wide information on protein numbers in Escherichia coli along with their cell-to-cell variability [6]. In total, 121 were classified as essential, while 894 were classified as non-essential.…”

“…To account for the stochasticity and the rate limiting steps of the underlying steps in the process of gene expression, we use the delayed stochastic modeling strategy [16] to drive the dynamics of the models, as it allows the use of non-Markovian dynamics to model the non-instantaneous processes underlying transcription and translation [17]. The parameters used in the models are extracted from live, single-cell, single-molecule measurements [6,13,14]. …”