RNA dynamics in live Escherichia coli cells

Abstract

We describe a method for tracking RNA molecules in Escherichia coli that is sensitive to single copies of mRNA, and, using the method, we find that individual molecules can be followed for many hours in living cells. We observe distinct characteristic dynamics of RNA molecules, all consistent with the known life history of RNA in prokaryotes: localized motion consistent with the Brownian motion of an RNA polymer tethered to its template DNA, free diffusion, and a few examples of polymer chain dynamics that appear to be a combination of chain fluctuation and chain elongation attributable to RNA transcription. We also quantify some of the dynamics, such as width of the displacement distribution, diffusion coefficient, chain elongation rate, and distribution of molecule numbers, and compare them with known biophysical parameters of the E. coli system.T he bacterium Escherichia coli may well be the most thoroughly characterized model biological system. Much of its metabolism serves as the basic paradigm for DNA replication, RNA transcription, protein synthesis, and gene regulation (1, 2).Recently, techniques have become available that allow us to study central problems in genome organization and expression in individual living cells (3, 4), rather than rely on the averaged properties of large populations. These studies have added to our knowledge in two main areas: the heterogeneity among cells in a supposedly homogeneous population (5-7) and the spatiotemporal organization of macromolecules in the bacterial cell (8). The subcellular location of a variety of different proteins involved in cell division, such as the MinCDE family (9), are now known to be localized in special regions, as is the location of replicating chromosomes (10) and several different plasmids (11,12). Complexes of signaling proteins in Bacillus subtilis and Caulobacter crescentus have likewise been localized to the poles of dividing and differentiating cells (8, 13).To our knowledge, there are no published studies on the localization and timing of mRNA synthesis in living bacterial cells. Our knowledge of RNA transcription and dynamics comes from population studies, or in vitro studies with purified components (14-17). A rare glimpse into cell-to-cell variability in message number was provided by the studies of with Salmonella using whole fixed mounts. However, there is no information at the single-cell level in living cells on message localization: whether mRNA is free to move throughout the cytoplasm, whether movement is active or passive, and, for RNA molecules present in low copy numbers, how the actual number of molecules is a function of cell state. This last information is of particular interest because it bears directly on the question of the regulation of proteins known to be present in low copy numbers (for example, many of the proteins that act to switch genes on and off) and how the cell successfully regulates copy number in the presence of considerable extrinsic and intrinsic noise (see ref. 7 for a review).Here we describe a metho...

Regulation of mean and noise of the in vivo kinetics of transcription under the control of the lac/ara‐1 promoter

“…coli strain DH5a-PRO (identical to DH5a-Z1) was generously provided by I. Golding of the University of Illinois, and contains two constructs: (i) PROTET-K133 carrying P LtetO-1 -MS2d-GFP [23], and (ii) a pIG-BAC (P lac/ara-1 -mRFP1-MS2-96bs) vector, carrying a 96 binding site array under the control of P lac/ara-1 [23].…”

“…When the reporter and target RNA are co-expressed, MS2d-GFP binds to the target RNA, forming a bright fluorescent spot. The RNA becomes visible during, or shortly after elongation [23]. Images of cells were taken from each slide every minute over two hours [7].…”

“…In these, the RNA is visible shortly after transcription occurs by tagging them with MS2d-GFP proteins [23]. By detecting when the target RNAs first appear, it was possible to measure the intervals between consecutive transcription events.…”

Real-time RNA profiling within a single bacterium

“…Given the transient character of RNA transcripts, it has proved technically challenging to monitor in real time the transcription activity of a promoter within an individual prokaryotic cell (2)(3)(4). There exist various powerful in vitro methods for measuring RNA levels, including Northern blots, RT-PCR, and microarrays.…”

Imaging Translation in Single Cells Using Fluorescent Microscopy

“…In bacteria, the counting of individual mRNAs in single cells showed that transcriptional bursting, infrequent transcription events that produce many transcripts within a short period of time, Fusco et al 2003;reprinted, with permission, from Elsevier # 2003. ) was responsible for the variability in gene expression (Golding and Cox 2004;Golding et al 2005). Similarly, in the eukaryote Dictyostelium, transcription of an endogenous developmental gene, dscA, was observed to be expressed in discrete bursts of gene activity (Chubb et al 2006).…”