The RelA-mediated stringent response is at the heart of bacterial adaptation to starvation and stress, playing a major role in the bacterial cell cycle and virulence. RelA integrates several environmental cues and synthesizes the alarmone ppGpp, which globally reprograms transcription, translation, and replication. We have developed and implemented novel single-molecule tracking methodology to characterize the intracellular catalytic cycle of RelA. Our single-molecule experiments show that RelA is on the ribosome under nonstarved conditions and that the individual enzyme molecule stays off the ribosome for an extended period of time after activation. This suggests that the catalytically active part of the RelA cycle is performed off, rather than on, the ribosome, and that rebinding to the ribosome is not necessary to trigger each ppGpp synthesis event. Furthermore, we find fast activation of RelA in response to heat stress followed by RelA rapidly being reset to its inactive state, which makes the system sensitive to new environmental cues and hints at an underlying excitable response mechanism. cytosolic diffusion | single particle tracking | photoactivated localization microscopy | stroboscopic illumination

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...

Single-Pair Fluorescence Resonance Energy Transfer (FRET) experiments reveal structural and dynamic information about macro-molecules by monitoring the change in FRET efficiency between fluorescent dyes attached to a macromolecule. The Nano-Positioning System (NPS) developed recently [1] uses data from several of such experiments to infer the position of a dye attached to protein sites unresolved by x-ray crystallography. Briefly, we perform probabilistic data analysis that allows us to calculate the distribution of possible dye positions in a simple and objective way without relying on ad-hoc procedures. Up to now NPS was limited to the triangulation of just one fluorescently labelled position based on FRET measurements to several other positions known from crystal structure [1,2]. Here, we discuss ways to extend the present model beyond this basic triangulation principle. In particular, we show how to gain three dimensional distance information by analysing triangulation networks where FRET is measured between arbitrary labelling sites in absence of other structural information.[1] A.

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The ribosome is one of the most important macromolecular complexes in a living cell. We have tracked individual 30S ribosomal subunits in exponentially growing E. coli cells using threedimensional single-particle tracking, where the z -position is estimated using astigmatism. The 30S subunits are stochiometrically labeled with S2-mEos2 by replacing the rpsB gene in the E. coli chromosome by rpsB-mEos2. The spatial precision in tracking is 20 nm in xy and 70 nm in z. The average trajectory consists of 4 steps corresponding to 80 ms. The trajectories are excluded from parts of the cell, consistent with nucleoid exclusion, and display isotropic diffusion with nearly identical apparent diffusion coefficients (0.05 ± 0.01 µm 2 s −1 ) in x, y and z. The tracking data fits well to a two-state diffusion model where 46% of the molecules are diffusing at 0.02 µm 2 s −1 and 54% are diffusing at 0.14 µm 2 s −1 . These states could correspond to translating 70S ribosomes and free 30S subunits.