Single-molecule FRET (smFRET) has become a mainstream technique for studying biomolecular structural dynamics The rapid and wide adoption of smFRET experiments by an ever-increasing number of groups has generated significant progress in sample preparation, measurement procedures, data analysis, algorithms and documentation Several labs that employ smFRET approaches have joined forces to inform the smFRET community about streamlining how to perform experiments and analyze results for obtaining quantitative information on biomolecular structure and dynamics The recent efforts include blind tests to assess the accuracy and the precision of smFRET experiments among different labs using various procedures These multi-lab studies have led to the development of smFRET procedures and documentation, which are important when submitting entries into the archiving system for integrative structure models, PDB-Dev This position paper describes the current 'state of the art' from different perspectives, points to unresolved methodological issues for quantitative structural studies, provides a set of 'soft recommendations' about which an emerging consensus exists, and lists openly available resources for newcomers and seasoned practitioners To make further progress, we strongly encourage 'open science' practices

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FRET-based dynamic structural biology: Challenges, perspectives and an appeal for open-science practices

In the past decades, advances in microscopy have made it possible to study the dynamics of individual biomolecules in vitro and resolve intramolecular kinetics that would otherwise be hidden in ensemble averages More recently, single-molecule methods have been used to image, localize, and track individually labeled macromolecules in the cytoplasm of living cells, allowing investigations of intermolecular kinetics under physiologically relevant conditions In this review, we illuminate the particular advantages of single-molecule techniques when studying kinetics in living cells and discuss solutions to specific challenges associated with these methods

Single-Molecule Kinetics in Living Cells.

Coupled transcription-translation (CTT) is a hallmark of prokaryotic gene expression. CTT occurs when ribosomes associate with and initiate translation of mRNAs whose transcription has not yet concluded, therefore forming "RNAP.mRNA.ribosome" complexes. CTT is a well-documented phenomenon that is involved in important gene regulation processes, such as attenuation and operon polarity. Despite the progress in our understanding of the cellular signals that coordinate CTT, certain aspects of its molecular architecture remain controversial. Additionally, new information on the spatial segregation between the transcriptional and the translational machineries in certain species, and on the capability of certain mRNAs to localize translation-independently, questions the unanimous occurrence of CTT. Furthermore, studies where transcription and translation were artificially uncoupled showed that transcription elongation can proceed in a translation-independent manner. Here, we review studies supporting the occurrence of CTT and findings questioning its extent, as well as discuss mechanisms that may explain both coupling and uncoupling, e.g., chromosome relocation and the involvement of cis- or trans-acting elements, such as small RNAs and RNA-binding proteins. These mechanisms impact RNA localization, stability, and translation. Understanding the two options by which genes can be expressed and their consequences should shed light on a new layer of control of bacterial transcripts fate.

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Coupled Transcription-Translation in Prokaryotes: An Old Couple With New Surprises.

Variational Algorithms for Analyzing Noisy Multistate Diffusion Trajectories

Abstract Translation is the fundamental process of protein synthesis and is catalysed by the ribosome in all living cells 1 . Here we use advances in cryo-electron tomography and sub-tomogram analysis 2,3 to visualize the structural dynamics of translation inside the bacterium Mycoplasma pneumoniae . To interpret the functional states in detail, we first obtain a high-resolution in-cell average map of all translating ribosomes and build an atomic model for the M. pneumoniae ribosome that reveals distinct extensions of ribosomal proteins. Classification then resolves 13 ribosome states that differ in their conformation and composition. These recapitulate major states that were previously resolved in vitro, and reflect intermediates during active translation. On the basis of these states, we animate translation elongation inside native cells and show how antibiotics reshape the cellular translation landscapes. During translation elongation, ribosomes often assemble in defined three-dimensional arrangements to form polysomes 4 . By mapping the intracellular organization of translating ribosomes, we show that their association into polysomes involves a local coordination mechanism that is mediated by the ribosomal protein L9. We propose that an extended conformation of L9 within polysomes mitigates collisions to facilitate translation fidelity. Our work thus demonstrates the feasibility of visualizing molecular processes at atomic detail inside cells. 

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Visualizing translation dynamics at atomic detail inside a bacterial cell

Our ability to directly relate results from test-tube biochemical experiments to the kinetics in living cells is very limited. Here we present experimental and analytical tools to directly study the kinetics of fast biochemical reactions in live cells. Dye-labeled molecules are electroporated into bacterial cells and tracked using super-resolved single-molecule microscopy. Trajectories are analyzed by machine-learning algorithms to directly monitor transitions between bound and free states. In particular, we measure the dwell time of tRNAs on ribosomes, and hence achieve direct measurements of translation rates inside living cells at codon resolution. We find elongation rates with tRNAPhe that are in perfect agreement with previous indirect estimates, and once fMet-tRNAfMet has bound to the 30S ribosomal subunit, initiation of translation is surprisingly fast and does not limit the overall rate of protein synthesis. The experimental and analytical tools for direct kinetics measurements in live cells have applications far beyond bacterial protein synthesis.

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tRNA tracking for direct measurements of protein synthesis kinetics in live 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.

https://www.pnas.org/content/pnas/118/9/e2013315118.full.pdf

Real-time measurements of aminoglycoside effects on protein synthesis in live cells

Abstract Many essential processes in the cell depend on proteins that use nucleoside triphosphates (NTPs). Methods that directly monitor the often-complex dynamics of these proteins at the single-molecule level have helped to uncover their mechanisms of action. However, the measurement throughput is typically limited for NTP-utilizing reactions, and the quantitative dissection of complex dynamics over multiple sequential turnovers remains challenging. Here we present a method for controlling NTP-driven reactions in single-molecule experiments via the local generation of NTPs (LAGOON) that markedly increases the measurement throughput and enables single-turnover observations. We demonstrate the effectiveness of LAGOON in single-molecule fluorescence and force spectroscopy assays by monitoring DNA unwinding, nucleosome sliding and RNA polymerase elongation. LAGOON can be readily integrated with many single-molecule techniques, and we anticipate that it will facilitate studies of a wide range of crucial NTP-driven processes. 

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Javier Aguirre Rivera

Papers

tRNA tracking for direct measurements of protein synthesis kinetics in live cells.

Real-time measurements of aminoglycoside effects on protein synthesis in live cells

Spatiotemporally controlled generation of NTPs for single-molecule studies

Direct measurements of erythromycin’s effect on protein synthesis kinetics in living bacterial cells