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Single-molecule analysis

A ribosome in action

The manufacture of proteins by ribosomes involves complex interactions of diverse nucleic-acid and protein ligands. Single-molecule studies allow us, for the first time, to follow the synthesis of full-length proteins in real time.

Protein synthesis involves a complex interplay of various cellular components. Ribosomes are the cell's protein-production factories, and interact with messenger RNA (the template), amino-acylated transfer RNAs (which act as adaptors between mRNA and amino-acid residues) and diverse co-factors (for the initiation of synthesis, elongation of the nascent chain and release of the mature polypeptide). In this issue, Uemura et al.1 report the use of an extremely sensitive single-molecule detection technique to observe this process at unprecedented resolution: the stepwise synthesis of a single protein (page 1012).

Ribosomes are evolutionarily conserved molecular nanomachines with a diameter of about 25 nanometres and a molecular weight of around 2.5 megadaltons. In functional terms, they are amino-acid polymerase enzymes with an RNA 'heart'. They accelerate the rate of protein synthesis by at least one millionfold, owing exclusively to entropic effects that involve the positioning of aminoacyl-tRNAs, the shielding of the reaction from bulk solvent and the organization of their own active site2,3. Ribosomes also check the quality of their polypeptide products — inaccurate amino-acid sequences could result in an altered three-dimensional protein structure and even cellular toxicity.

Structural analyses of functional ribosome complexes have formed the basis of a consistent biochemical model for the mechanism of protein synthesis4, suggesting that this process depends on large-scale conformational changes in the ribosome. But the nature, timescale and magnitude of these dynamic changes have remained undefined.

Traditional biophysical investigations of dynamic molecular processes yield ensemble-averaged data. These may hide crucial information owing to the presence of asynchronous conformational states and varying stages of the enzymatic reaction. By contrast, single-molecule studies circumvent the need for synchronous molecular behaviour and the use of uniform samples, thus allowing the identification and direct characterization of transient, rare and so physiologically relevant events. With the use of sensitive fluorophores, single-molecule spectroscopy and force-based techniques, researchers can observe changes in distance on the subnanometre scale with a temporal resolution in the millisecond range.

To study the details of translation, for example, fluorophores can be chemically coupled to tRNA molecules. Consequently, after binding to the ribosome, the fluorescently labelled tRNAs 'report' with high sensitivity on how they are selected by the ribosome, their motion within the ribosome and even conformational changes in the ribosome.

Indeed, single-molecule analyses of fluorescently labelled tRNAs have allowed the monitoring of tRNA selection and (single) peptide-bond formation in real time, as well as studies of tRNA dynamics on the ribosome during elongation, and single-molecule force measurements have helped to determine the strength of ribosome–mRNA interactions5. Although such studies have revealed previously unknown details of tRNA selection and catalytically important ribosomal states, the findings were limited to the initial stages of translation. A desirable extension of this is the investigation of several rounds of peptide-bond formation — for instance, study of the continuity of synthesis (processivity) or its accuracy (translational fidelity).

The first study to attempt this6 used specifically designed, single hairpin-shaped mRNA sequences tethered by their ends to optical tweezers, and determined their translocation along the ribosome. The translocation measured three mRNA bases (one codon) and occurred as a series of movement–pause–movement events, with pause durations ranging from a fraction of a second to several seconds. The 'dwell' time at each codon was the time taken for tRNA selection and peptide-bond formation, which can be correlated to the sequence context.

Uemura and colleagues1 now take these methods even further. Their approach is based on a set of tRNAs labelled with distinct fluorophores, allowing their immediate identification after ribosomal binding and so real-time 'reading' of the underlying mRNA sequence. By watching tRNA substrates instead of the template mRNA, the authors were able to follow tRNA binding and transit on individual translating ribosomes during multiple rounds of chain elongation in the synthesis of short peptides (4–13 amino acids long). Moreover, they showed that only two tRNA molecules simultaneously bind to the ribosome, allowing them to explore the mechanism of tRNA transit in relation to other reactants (Fig. 1).

Figure 1: Close-up view of a zero-mode waveguide vessel.
figure1

Uemura and colleagues' set-up1 consisted of a nanostructured metal film called a zero-mode waveguide, containing circular holes of diameter 50–200 nanometres. Using this, they analysed sample volumes as small as 10−21 litres to detect individual fluorescent tRNA molecules against the background signal from freely diffusing tRNA molecules. The binding of fluorescently labelled, cognate tRNA molecules — each labelled with a different fluorophore — within the ribosomal active site causes an elevation in fluorescent emission on the corresponding colour channel.

What is of utmost importance is that Uemura et al. were able to study translation at physiologically relevant (micromolar) concentrations of tRNA and other factors using extremely small reaction vessels known as zero-mode waveguides, which were generated from nanophotonic metal films and illuminated from below by a laser. The resulting observation volumes of 10−21 litres were therefore occupied by, on average, not more than one molecule (Fig. 1). Because the duration of binding of the correct fluorescently labelled tRNA to the ribosome was much longer than the timescales associated with the freely diffusing tRNA molecules, genuine signals were reliably distinguished from low and constant background.

The real-time monitoring of single translating ribosomes is truly a breakthrough for studying the process of translation, how its accuracy is controlled and the conformational changes that it involves in ribosomes. It should now be only a small step to elucidating the correlation between tRNA binding and changes in ribosomal conformation, to probing the dynamics of translational control, and to studying the synthesis and folding of full-length proteins in real time. Beyond that, Uemura and colleagues' impressive demonstration1 of how their powerful technique (originally developed for 'next-generation' DNA sequencing7) can be used to answer biological questions will play a decisive part in studying other processes, including RNA sequencing, the sequencing of genomic sites modified by methyl groups, and various time-dependent regulatory processes.

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Brakmann, S. A ribosome in action. Nature 464, 987–988 (2010). https://doi.org/10.1038/464987a

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