Logo: to the web site of Uppsala University

Single-molecule tracking (SMT) is a powerful tool for real-time studies of protein interactions in living cells. Dye-labelled SNAP-tag and HaloTag self-labelling proteins have simplified SMT significantly, due to their superior photophysical properties compared to fluorescent proteins. However, due to their size, fusion of these tags to a protein of interest often results in loss of protein function. We introduce FLORENCE – a universal labelling method for SMT, based on genetic code expansion (GCE). We overcome significant caveats related to re-coded strains, vectors, and dyes and report successful tracking of site-specifically intracellularly labelled proteins in genomically re-coded E. coli. Our findings establish a robust in vivo protein-labelling strategy, expanding the capabilities of SMT as a method to study the dynamics of proteins in living cells. Moreover, we observe that the strain-promoted azide–alkyne click-chemistry reaction occurs as fast as 30 min in live E. coli cells and can be used as a robust labelling reaction.

The main function of translation initiation factors is to assist ribosomes in selecting the correct reading frame on an mRNA. This process has been extensively studied with the help of reconstituted in vitro systems, but the dynamics in living cells have not been characterized. In this study, we performed single-molecule tracking of the bacterial initiation factors IF2, IF3, as well as the initiator fMet-tRNA^fMet directly in growing Escherichia coli cells. Our results reveal the kinetics of factor association with the ribosome and, among other things, highlight the respective antagonistic roles of IF2 and IF3 in the process. Importantly, our comparisons of in vivo binding kinetics of two naturally occurring isoforms of IF2 reveal that the longer IF2α isoform directly interacts with the transcriptional factor NusA, a finding further corroborated by pull-down and cross-linking experiments. Our results suggest that this interaction may promote formation of a coupled transcription-translation complex early in the translation cycle, motivating further structural studies to validate the mechanism. We further show that cells with compromised binding between IF2α and NusA display slow adaptation to new growth conditions.

Electroporation of dye-labeled bio-molecules into bacteria has proven to be a valuable route for single-molecule tracking in living cells. However, control over cell viability, electroporation efficiency, and environment conditions before, during, and after electroporation is difficult to achieve in bulk experiments. Here, a microfluidic platform is presented capable of single-cell electroporation with in situ microscopy and demonstrate delivery of DNA into bacteria. Via real time observation of the electroporation process, it is found that the effect of electrophoresis plays an important role when performing electroporation in a miniaturized platform and show that its undesired action can be balanced by using bipolar electrical pulses. It is suggested that a low temperature of the sample during electroporation is important for cell viability due to temperature-dependant viscoelastic properties of the cell membrane. It is further found that the presence of low conductive liquid between cells and the electrodes leads to a voltage divider effect that strongly influences the success of on-chip electroporation. Finally, it is concluded that electroporation is a highly stochastic process and envision that the microfluidic system presented here, capable of single-cell read-out, can be used for further fundamental studies to increase the understanding of the electroporation process in bacterial cells.

The bacterial chaperone Trigger factor (TF) binds to ribosome-nascent chain complexes (RNCs) and cotranslationally aids the folding of proteins in bacteria. Decades of studies have given a broad, but often conflicting, description of the substrate specificity of TF, its RNC-binding dynamics, and competition with other RNC-binding factors, such as the Signal Recognition Particle (SRP). Previous RNC-binding kinetics experiments were commonly conducted on stalled RNCs in reconstituted systems, and consequently, may not be representative of the interaction of TF with ribosomes translating mRNA in the cytoplasm of the cell. Here, we used single-particle tracking (SPT) to measure TF binding to actively translating ribosomes inside living Escherichia coli. In cells, TF displays distinct binding modes—longer (ca 1 s) and shorter (ca 50 ms) RNC bindings. Consequently, we conclude that TF, on average, stays bound to the RNC for only a fraction of the translation cycle. Further, binding events are interrupted only by transient excursions to a freely diffusing state (ca 40 ms), suggesting a highly dynamic binding and unbinding cycle of TF in vivo. We also show that TF competes with SRP for RNC binding, and in doing so, tunes the binding selectivity of SRP.

Reaching sub-millisecond 3D tracking of individual molecules in living cells would enable direct measurements of diffusion-limited macromolecular interactions under physiological conditions. Here, we present a 3D tracking principle that approaches the relevant regime. The method is based on the true excitation point spread function and cross-entropy minimization for position localization of moving fluorescent reporters. Tests on beads moving on a stage reaches 67 nm lateral and 109 nm axial precision with a time resolution of 0.84 ms at a photon count rate of 60 kHz; the measurements agree with the theoretical and simulated predictions. Our implementation also features a method for microsecond 3D PSF positioning and an estimator for diffusion analysis of tracking data. Finally, we successfully apply these methods to track the Trigger Factor protein in living bacterial cells. Overall, our results show that while it is possible to reach sub-millisecond live-cell single-molecule tracking, it is still hard to resolve state transitions based on diffusivity at this time scale.

Metelev et al. use single-molecule tracking to study kinetics of translation directly in E. coli cells, and how it is affected by translation inhibitors and rRNA mutations. Their results support widespread 70S re-initiation on mRNAs. Ribosome mediated mRNA translation is central to life. The cycle of translation, however, has been characterized mostly using reconstituted systems, with only few techniques applicable for studies in the living cell. Here we describe a live-cell ribosome-labeling method, which allows us to characterize the whole processes of finding and translating an mRNA, using single-molecule tracking techniques. We find that more than 90% of both bacterial ribosomal subunits are engaged in translation at any particular time, and that the 30S and 50S ribosomal subunits spend the same average time bound to an mRNA, revealing that 30S re-initiation on poly-cistronic mRNAs is not prevalent in E. coli. Instead, our results are best explained by substantial 70S re-initiation of translation of poly-cistronic mRNAs, which is further corroborated by experiments with translation initiation inhibitors. Finally, we find that a variety of previously described orthogonal ribosomes, with altered anti-Shine-Dalgarno sequences, show significant binding to endogenous mRNAs.

Mechanistic details of the signal recognition particle (SRP)-mediated insertion of membrane proteins have been described from decades of in vitro biochemical studies. However, the dynamics of the pathway inside the living cell remain obscure. By combining in vivo single-molecule tracking with numerical modeling and simulated microscopy, we have constructed a quantitative reaction-diffusion model of the SRP cycle. Our results suggest that the SRP-ribosome complex finds its target, the membrane-bound translocon, through a combination of three-dimensional (3D) and 2D diffusional search, together taking on average 750 ms. During this time, the nascent peptide is expected to be elongated only 12 or 13 amino acids, which explains why, in Escherichia coli, no translation arrest is needed to prevent incorrect folding of the polypeptide in the cytosol. We also found that a remarkably high proportion (75%) of SRP bindings to ribosomes occur in the cytosol, suggesting that the majority of target ribosomes bind SRP before reaching the membrane. In combination with the average SRP cycling time, 2.2 s, this result further shows that the SRP pathway is capable of targeting all substrate ribosomes to translocons.

Macrolide antibiotics, such as erythromycin, bind to the nascent peptide exit tunnel (NPET) of the bacterial ribosome and modulate protein synthesis depending on the nascent peptide sequence. Whereas in vitro biochemical and structural methods have been instrumental in dissecting and explaining the molecular details of macrolide-induced peptidyl-tRNA drop-off and ribosome stalling, the dynamic effects of the drugs on ongoing protein synthesis inside live bacterial cells are far less explored. In the present study, we used single-particle tracking of dye-labeled tRNAs to study the kinetics of mRNA translation in the presence of erythromycin, directly inside live Escherichia coli cells. In erythromycin-treated cells, we find that the dwells of elongator tRNA(Phe) on ribosomes extend significantly, but they occur much more seldom. In contrast, the drug barely affects the ribosome binding events of the initiator tRNA(fMet). By overexpressing specific short peptides, we further find context-specific ribosome binding dynamics of tRNA(Phe), underscoring the complexity of erythromycin's effect on protein synthesis in bacterial cells.

The spread of antibiotic resistance is turning many of the currently used antibiotics less effective against common infections. To address this public health challenge, it is critical to enhance our understanding of the mechanisms of action of these compounds. Aminoglycoside drugs bind the bacterial ribosome, and decades of results from in vitro biochemical and structural approaches suggest that these drugs disrupt protein synthesis by inhibiting the ribosome's translocation on the messenger RNA, as well as by inducing miscoding errors. So far, however, we have sparse information about the dynamic effects of these compounds on protein synthesis inside the cell. In the present study, we measured the effect of the aminoglycosides apramycin, gentamicin, and paromomycin on ongoing protein synthesis directly in live Escherichia coli cells by tracking the binding of dye-labeled transfer RNAs to ribosomes. Our results suggest that the drugs slow down translation elongation two- to fourfold in general, and the number of elongation cycles per initiation event seems to decrease to the same extent. Hence, our results imply that none of the drugs used in this study cause severe inhibition of translocation.

Many proteins that bind specific DNA sequences search the genome by combining three-dimensional diffusion with one-dimensional sliding on nonspecific DNA(1-5). Here we combine resonance energy transfer and fluorescence correlation measurements to characterize how individual lac repressor (LacI) molecules explore the DNA surface during the one-dimensional phase of target search. To track the rotation of sliding LacI molecules on the microsecond timescale, we use real-time single-molecule confocal laser tracking combined with fluorescence correlation spectroscopy (SMCT-FCS). The fluctuations in fluorescence signal are accurately described by rotation-coupled sliding, in which LacI traverses about 40 base pairs (bp) per revolution. This distance substantially exceeds the 10.5-bp helical pitch of DNA; this suggests that the sliding protein frequently hops out of the DNA groove, which would result in the frequent bypassing of target sequences. We directly observe such bypassing using single-molecule fluorescence resonance energy transfer (smFRET). A combined analysis of the smFRET and SMCT-FCS data shows that LacI hops one or two grooves (10-20 bp) every 200-700 mu s. Our data suggest a trade-off between speed and accuracy during sliding: the weak nature of nonspecific protein-DNA interactions underlies operator bypassing, but also speeds up sliding. We anticipate that SMCT-FCS, which monitors rotational diffusion on the microsecond timescale while tracking individual molecules with millisecond resolution, will be applicable to the real-time investigation of many other biological interactions and will effectively extend the accessible time regime for observing these interactions by two orders of magnitude. Single-molecule fluorescence resonance energy transfer and real-time confocal laser tracking with fluorescence correlation spectroscopy together characterize how individual lac repressor molecules bypass operator sites while exploring the DNA surface at microsecond timescales.