A new approach to synthetic chemistry is performed in ultraminiaturized, nanofabricated reaction chambers. Using lithographically defined nanowells, we achieve single-point covalent chemistry on hundreds of individual carbon nanotube transistors, providing robust statistics and unprecedented spatial resolution in adduct position. Each device acts as a sensor to detect, in real-time and through quantized changes in conductance, single-point functionalization of the nanotube as well as consecutive chemical reactions, molecular interactions, and molecular conformational changes occurring on the resulting single-molecule probe. In particular, we use a set of sequential bioconjugation reactions to tether a single-strand of DNA to the device and record its repeated, reversible folding into a G-quadruplex structure. The stable covalent tether allows us to measure the same molecule in different solutions, revealing the characteristic increased stability of the G-quadruplex structure in the presence of potassium ions (K+) versus sodium ions (Na+). Nanowell-confined reaction chemistry on carbon nanotube devices offers a versatile method to isolate and monitor individual molecules during successive chemical reactions over an extended period of time.

The cellular translational machinery (TM) synthesizes proteins using exclusively L- or achiral aminoacyl-tRNAs (aa-tRNAs), despite the presence of D-amino acids in nature and their ability to be aminoacylated onto tRNAs by aa-tRNA synthetases. The ubiquity of L-amino acids in proteins has led to the hypothesis that D-amino acids are not substrates for the TM. Supporting this view, protein engineering efforts to incorporate D-amino acids into proteins using the TM have thus far been unsuccessful. Nonetheless, a mechanistic understanding of why D-aa-tRNAs are poor substrates for the TM is lacking. To address this deficiency, we have systematically tested the translation activity of D-aa-tRNAs using a series of biochemical assays. We find that the TM can effectively, albeit slowly, accept D-aa-tRNAs into the ribosomal aa-tRNA binding (A) site, use the A-site D-aa-tRNA as a peptidyl-transfer acceptor, and translocate the resulting peptidyl-D-aa-tRNA into the ribosomal peptidyl-tRNA binding (P) site. During the next round of continuous translation, however, we find that ribosomes carrying a P-site peptidyl-D-aa-tRNA partition into subpopulations that are either translationally arrested or that can continue translating. Consistent with its ability to arrest translation, chemical protection experiments and molecular dynamics simulations show that P site-bound peptidyl-D-aa-tRNA can trap the ribosomal peptidyl-transferase center in a conformation in which peptidyl transfer is impaired. Our results reveal a novel mechanism through which D-aa-tRNAs interfere with translation, provide insight into how the TM might be engineered to use D-aa-tRNAs, and increase our understanding of the physiological role of a widely distributed enzyme that clears D-aa-tRNAs from cells.

One of the most challenging unanswered questions regarding the structural biology of biomolecular machines such as the two-subunit ribosome is whether and how these machines coordinate seemingly independent and random conformational fluctuations to maximize and regulate their functional efficiencies. To address this question, we have used ribosome mutagenesis or a ribosome-targeting antibiotic to predictably perturb the dynamics of intersubunit rotation, a structural rearrangement of the ribosome that is essential for the translocation and ejection of ribosome-bound tRNAs during translation. Concomitantly, we have used single-molecule fluorescence resonance energy transfer (smFRET) to characterize the effects of these perturbations on the dynamics of ribosomal L1 stalk movements and ribosome-bound tRNA reconfigurations, conformational changes that are likewise essential for the translocation and ejection of tRNAs during translation. Together with the results of complementary biochemical studies, our smFRET studies demonstrate that the ribosome uses cooperative conformational changes to maximize and regulate the efficiency with which it translocates and ejects tRNAs during translation. We propose that the ribosome employs cooperative conformational changes to efficiently populate global conformational states that are productive for translation, that translation factors exploit this cooperativity as part of their mechanisms of action, and that antibiotics exploit it to maximize the potency with which they inhibit translation. It is likely that similar cooperative conformational changes underlie the function and regulation of other biomolecular machines.

The optical confinement generated by metal-based nanoapertures fabricated on a silica substrate has recently enabled single-molecule fluorescence measurements to be performed at physiologically relevant background concentrations of fluorophore-labeled biomolecules. Nonspecific adsorption of fluorophore-labeled biomolecules to the metallic cladding and silica bottoms of nanoapertures, however, remains a critical limitation. To overcome this limitation, we have developed a selective functionalization chemistry whereby the metallic cladding of gold nanoaperture arrays is passivated with methoxy-terminated, thiol-derivatized polyethylene glycol (PEG), and the silica bottoms of those arrays are functionalized with a binary mixture of methoxy- and biotin-terminated, silane-derivatized PEG. This functionalization scheme enables biotinylated target biomolecules to be selectively tethered to the silica nanoaperture bottoms via biotin–streptavidin interactions and reduces the nonspecific adsorption of fluorophore-labeled ligand biomolecules. This, in turn, enables the observation of ligand biomolecules binding to their target biomolecules even under greater than 1 μM background concentrations of ligand biomolecules, thereby rendering previously impracticable biological systems accessible to single-molecule fluorescence investigations.Keywords: fluorescence resonance energy transfer; microscopy; nanoaperture; self-assembled monolayer; single-molecule fluorescence; translation factor; zero-mode waveguide

Determining the mechanism by which tRNAs rapidly and precisely transit through the ribosomal A, P, and E sites during translation remains a major goal in the study of protein synthesis. Here, we report the real-time dynamics of the L1 stalk, a structural element of the large ribosomal subunit that is implicated in directing tRNA movements during translation. Within pretranslocation ribosomal complexes, the L1 stalk exists in a dynamic equilibrium between open and closed conformations. Binding of elongation factor G (EF-G) shifts this equilibrium toward the closed conformation through one of at least two distinct kinetic mechanisms, where the identity of the P-site tRNA dictates the kinetic route that is taken. Within posttranslocation complexes, L1 stalk dynamics are dependent on the presence and identity of the E-site tRNA. Collectively, our data demonstrate that EF-G and the L1 stalk allosterically collaborate to direct tRNA translocation from the P to the E sites, and suggest a model for the release of E-site tRNA.

By using single-molecule fluorescence resonance energy transfer (smFRET), we observe the real-time dynamic coupling between the ribosome, labeled at the L1 stalk, and transfer RNA (tRNA). We find that an interaction between the ribosomal L1 stalk and the newly deacylated tRNA is established spontaneously upon peptide bond formation; this event involves coupled movements of the L1 stalk and tRNAs as well as ratcheting of the ribosome. In the absence of elongation factor G, the entire pretranslocation ribosome fluctuates between just two states: a nonratcheted state, with tRNAs in their classical configuration and no L1 stalk-tRNA interaction, and a ratcheted state, with tRNAs in an intermediate hybrid configuration and a direct L1 stalk-tRNA interaction. We demonstrate that binding of EF-G shifts the equilibrium toward the ratcheted state. Real-time smFRET experiments reveal that the L1 stalk-tRNA interaction persists throughout the translocation reaction, suggesting that the L1 stalk acts to direct tRNA movements during translocation.

•IF2 catalyzes ribosomal 50S subunit joining to the ribosomal 30S IC through the formation of a 30S IC-bound IF2·tRNA sub-complex.•We have developed a novel IF2·tRNA single-molecule fluorescence resonance energy transfer signal to monitor the stability and dynamics of the IF2·tRNA sub-complex on the 30S IC.•We show that IF2 binds stably to 30S ICs that are associated with rapid subunit joining and that the IF2·tRNA sub-complex in these 30S ICs favors a single conformation.•Additionally, we show that IF2 either binds less stably to or forms an IF2·tRNA sub-complex with altered dynamics on 30S ICs associated with slower subunit joining.•We conclude that the stability of IF2 and the conformational dynamics of the IF2·tRNA sub-complex can be tuned in order to regulate the rate of subunit joining.Joining of the large, 50S, ribosomal subunit to the small, 30S, ribosomal subunit initiation complex (IC) during bacterial translation initiation is catalyzed by the initiation factor (IF) IF2. Because the rate of subunit joining is coupled to the IF, transfer RNA (tRNA), and mRNA codon compositions of the 30S IC, the subunit joining reaction functions as a kinetic checkpoint that regulates the fidelity of translation initiation. Recent structural studies suggest that the conformational dynamics of the IF2·tRNA sub-complex forming on the intersubunit surface of the 30S IC may play a significant role in the mechanisms that couple the rate of subunit joining to the IF, tRNA, and codon compositions of the 30S IC. To test this hypothesis, we have developed a single-molecule fluorescence resonance energy transfer signal between IF2 and tRNA that has enabled us to monitor the conformational dynamics of the IF2·tRNA sub-complex across a series of 30S ICs. Our results demonstrate that 30S ICs undergoing rapid subunit joining display a high affinity for IF2 and an IF2·tRNA sub-complex that primarily samples a single conformation. In contrast, 30S ICs that undergo slower subunit joining exhibit a decreased affinity for IF2 and/or a change in the conformational dynamics of the IF2·tRNA sub-complex. These results strongly suggest that 30S IC-driven changes in the stability of IF2 and the conformational dynamics of the IF2·tRNA sub-complex regulate the efficiency and fidelity of subunit joining during translation initiation.Download high-res image (187KB)Download full-size image

•Joining of the 50S subunit to the 30S IC during bacterial translation initiation is regulated by three protein IFs.•An smFRET assay was developed to probe interactions between IF2 and the GAC of the 50S subunit during subunit joining.•30S IC-bound IF3 imparts reversibility to IF2-mediated subunit joining and influences interactions between IF2 and the GAC.•Discrete modes of subunit joining were identified that resulted in 70S ICs with different lifetimes and conformations.•IF3 modulates the intersubunit contacts formed during subunit joining, likely by regulating the conformation of the 30S IC.Ribosomal subunit joining is a key checkpoint in the bacterial translation initiation pathway during which initiation factors (IFs) regulate association of the 30S initiation complex (IC) with the 50S subunit to control formation of a 70S IC that can enter into the elongation stage of protein synthesis. The GTP-bound form of IF2 accelerates subunit joining, whereas IF3 antagonizes subunit joining and plays a prominent role in maintaining translation initiation fidelity. The molecular mechanisms through which IF2 and IF3 collaborate to regulate the efficiency of 70S IC formation, including how they affect the dynamics of subunit joining, remain poorly defined. Here, we use single-molecule fluorescence resonance energy transfer to monitor the interactions between IF2 and the GTPase-associated center (GAC) of the 50S subunit during real-time subunit joining reactions in the absence and presence of IF3. In the presence of IF3, IF2-mediated subunit joining becomes reversible, and subunit joining events cluster into two distinct classes corresponding to formation of shorter- and longer-lifetime 70S ICs. Inclusion of IF3 within the 30S IC was also found to alter the conformation of IF2 relative to the GAC, suggesting that IF3's regulatory effects may stem in part from allosteric modulation of IF2–GAC interactions. The results are consistent with a model in which IF3 can exert control over the efficiency of subunit joining by modulating the conformation of the 30S IC, which in turn influences the formation of stabilizing intersubunit contacts and thus the reaction's degree of reversibility.Download high-res image (107KB)Download full-size image

Time series data provided by single-molecule Förster resonance energy transfer (smFRET) experiments offer the opportunity to infer not only model parameters describing molecular complexes, e.g., rate constants, but also information about the model itself, e.g., the number of conformational states. Resolving whether such states exist or how many of them exist requires a careful approach to the problem of model selection, here meaning discrimination among models with differing numbers of states. The most straightforward approach to model selection generalizes the common idea of maximum likelihood—selecting the most likely parameter values—to maximum evidence: selecting the most likely model. In either case, such an inference presents a tremendous computational challenge, which we here address by exploiting an approximation technique termed variational Bayesian expectation maximization. We demonstrate how this technique can be applied to temporal data such as smFRET time series; show superior statistical consistency relative to the maximum likelihood approach; compare its performance on smFRET data generated from experiments on the ribosome; and illustrate how model selection in such probabilistic or generative modeling can facilitate analysis of closely related temporal data currently prevalent in biophysics. Source code used in this analysis, including a graphical user interface, is available open source via http://vbFRET.sourceforge.net.

The selectivity with which a biomolecule can bind its cognate ligand when confronted by the vast array of structurally similar, competing ligands that are present in the cell underlies the fidelity of some of the most fundamental processes in biology. Because they collectively comprise one of only a few methods that can sensitively detect the ‘encounter’ complexes and subsequent intermediate states that regulate the selectivity of ligand binding, single-molecule fluorescence, and particularly single-molecule fluorescence resonance energy transfer (smFRET), approaches have revolutionized studies of ligand-binding reactions. Here, we describe a widely used smFRET strategy that enables investigations of a large variety of ligand-binding reactions, and discuss two such reactions, aminoacyl-tRNA selection during translation elongation and splice site selection during spliceosome assembly, that highlight both the successes and challenges of smFRET studies of ligand-binding reactions. We conclude by reviewing a number of emerging experimental and computational approaches that are expanding the capabilities of smFRET approaches for studies of ligand-binding reactions and that promise to reveal the mechanisms that control the selectivity of ligand binding with unprecedented resolution.