Single-particle tracking reveals that free ribosomal subunits are not excluded from the Escherichia coli nucleoid

Abstract

Biochemical and genetic data show that ribosomes closely follow RNA polymerases that are transcribing protein-coding genes in bacteria. At the same time, electron and fluorescence microscopy have revealed that ribosomes are excluded from the Escherichia coli nucleoid, which seems to be inconsistent with fast translation initiation on nascent mRNA transcripts. The apparent paradox can be reconciled if translation of nascent mRNAs can start throughout the nucleoid before they relocate to the periphery. However, this mechanism requires that free ribosomal subunits are not excluded from the nucleoid. Here, we use single-particle tracking in living E. coli cells to determine the fractions of free ribosomal subunits, classify individual subunits as free or mRNA-bound, and quantify the degree of exclusion of bound and free subunits separately. We show that free subunits are not excluded from the nucleoid. This finding strongly suggests that translation of nascent mRNAs can start throughout the nucleoid, which reconciles the spatial separation of DNA and ribosomes with cotranscriptional translation. We also show that, after translation inhibition, free subunit precursors are partially excluded from the compacted nucleoid. This finding indicates that it is active translation that normally allows ribosomal subunits to assemble on nascent mRNAs throughout the nucleoid and that the effects of translation inhibitors are enhanced by the limited access of ribosomal subunits to nascent mRNAs in the compacted nucleoid.nucleoid exclusion | transcription-translation coupling | antibiotics | single-molecule tracking | single-molecule imaging I n bacteria, translation often starts soon after the ribosomebinding site emerges from the RNA exit channel of the RNA polymerase. The transcribing RNA polymerase is then closely followed by translating ribosomes in such a way that the overall transcription elongation rate is tightly controlled by the translation rate (1). This coupling between transcription and translation of nascent mRNAs is important for regulatory mechanisms that respond to the formation of gaps between the transcribing RNA polymerases and the trailing ribosomes. Such gaps may, for example, allow the formation of secondary structures that allow RNA polymerases to proceed through transcription termination sites (2). The gaps may also allow the transcription termination factor Rho to access the nascent mRNAs and terminate transcription (3).Bacterial 70S ribosomes are formed when large 50S subunits and small 30S subunits assemble on mRNAs. Electron and fluorescence microscopy have revealed that ribosomes are excluded from the Escherichia coli nucleoid (4-6), but this spatial separation of DNA and ribosomes has not yet been reconciled with cotranscriptional translation. The paradox can be resolved if translation of nascent mRNAs can start throughout the nucleoid before they relocate to the periphery (7). However, this mechanism requires that free ribosomal subunits are not excluded from the nucleoid.To determine whether free r...

Robustness of the Process of Nucleoid Exclusion of Protein Aggregates in Escherichia coli

“…For the range of aggregate sizes detectable from the images, we were unable to find tangible differences between the spatial distributions of small-and normal-sized aggregates regardless of the nucleoid size. Nevertheless, we expect that below a certain aggregate size (beyond our detection range), nucleoid exclusion will lose much of its effectiveness, particularly given recent evidence that while ribosomes are excluded from the nucleoid, ribosomal subunits are not (43). In this regard, recent studies provided evidence that the aggregation of nonfunctional proteins is an active process (14).…”

Live-cell superresolution microscopy reveals the organization of RNA polymerase in the bacterial nucleoid

“…Defining the periphery as the exterior 50% of the cell width (yellow highlighted areas, Fig. 2F) gave only 29% of HU molecules in this region compared with 39% expected from a uniform distribution in a cylindrical cell volume (32). Performing the same analysis on mobile RNAP molecules showed that they matched extremely well to the distribution of HU, and also have 29% located in the cell periphery rather than uniformly filling the cell volume.…”

Applications of Microfluidics in Quantitative Biology

“…[26,27] They also showed that only translating ribosomes were excluded whereas free ribosomes could access the nucleoid freely. [28] Microfluidics were also explored as in vitro platforms in characterizing the transcription factor binding sites and the interactions between transcription factors. [29,30] Microfluidics has assisted in monitoring and quantifying long term cell behavior.…”