Ian Farrell

Dynamics of translation by single ribosomes through mRNA secondary structures

During protein synthesis, the ribosome translates nucleotide triplets in single-stranded mRNA into polypeptide sequences. Strong downstream mRNA secondary structures, which must be unfolded for translation, can slow or even halt protein synthesis. Here we used single-molecule fluorescence resonance energy transfer to determine reaction rates for specific steps within the elongation cycle as the Escherichia coli ribosome encounters stem-loop or pseudoknot mRNA secondary structures. Downstream stem-loops containing 100% GC base pairs decrease the rates of both tRNA translocation within the ribosome and deacylated tRNA dissociation from the ribosomal exit site (E site). Downstream stem-loops or pseudoknots containing both GC and AU pairs also decrease the rate of tRNA dissociation, but they have little effect on tRNA translocation rate. Thus, somewhat unexpectedly, unfolding of mRNA secondary structures is more closely coupled to E-site tRNA dissociation than to tRNA translocation.

FRET-Based Identification of mRNAs Undergoing Translation

We present proof-of-concept in vitro results demonstrating the feasibility of using single molecule fluorescence resonance energy transfer (smFRET) measurements to distinguish, in real time, between individual ribosomes programmed with several different, short mRNAs. For these measurements we use either the FRET signal generated between two tRNAs labeled with different fluorophores bound simultaneously in adjacent sites to the ribosome (tRNA-tRNA FRET) or the FRET signal generated between a labeled tRNA bound to the ribosome and a fluorescent derivative of ribosomal protein L1 (L1-tRNA FRET). With either technique, criteria were developed to identify the mRNAs, taking into account the relative activity of the mRNAs. These criteria enabled identification of the mRNA being translated by a given ribosome to within 95% confidence intervals based on the number of identified FRET traces. To upgrade the approach for natural mRNAs or more complex mixtures, the stoichiometry of labeling should be enhanced and photobleaching reduced. The potential for porting these methods into living cells is discussed.

Elongation factor G initiates translocation through a power stroke

Significance Elongation factor G (EF-G) uses energy stored in GTP to catalyze movement of transfer RNAs and messenger RNA in the ribosome during the translocation step of prokaryotic protein synthesis. Using single-molecule polarized fluorescence microscopy, three-dimensional rotational motions of individual domains of EF-G were directly captured, for the first time to our knowledge, during normal translocation. Our observations strongly imply a hybrid model, in which the initial steps of translocation are ribosome unlocking driven by a force generated via EF-G–dependent GTP hydrolysis, and further steps of translocation are mainly driven by the energetics of the ribosome itself. These results demonstrate that the ribosome and EF-G make use of power-stroke and Brownian-ratchet mechanisms to ensure efficiency and accuracy of translocation. During the translocation step of prokaryotic protein synthesis, elongation factor G (EF-G), a guanosine triphosphatase (GTPase), binds to the ribosomal PRE-translocation (PRE) complex and facilitates movement of transfer RNAs (tRNAs) and messenger RNA (mRNA) by one codon. Energy liberated by EF-G’s GTPase activity is necessary for EF-G to catalyze rapid and precise translocation. Whether this energy is used mainly to drive movements of the tRNAs and mRNA or to foster EF-G dissociation from the ribosome after translocation has been a long-lasting debate. Free EF-G, not bound to the ribosome, adopts quite different structures in its GTP and GDP forms. Structures of EF-G on the ribosome have been visualized at various intermediate steps along the translocation pathway, using antibiotics and nonhydolyzable GTP analogs to block translocation and to prolong the dwell time of EF-G on the ribosome. However, the structural dynamics of EF-G bound to the ribosome have not yet been described during normal, uninhibited translocation. Here, we report the rotational motions of EF-G domains during normal translocation detected by single-molecule polarized total internal reflection fluorescence (polTIRF) microscopy. Our study shows that EF-G has a small (∼10°) global rotational motion relative to the ribosome after GTP hydrolysis that exerts a force to unlock the ribosome. This is followed by a larger rotation within domain III of EF-G before its dissociation from the ribosome.

In vivo single-RNA tracking shows that most tRNA diffuses freely in live bacteria

Transfer RNA (tRNA) links messenger RNA nucleotide sequence with amino acid sequence during protein synthesis. Despite the importance of tRNA for translation, its subcellular distribution and diffusion properties in live cells are poorly understood. Here, we provide the first direct report on tRNA diffusion localization in live bacteria. We internalized tRNA labeled with organic fluorophores into live bacteria, applied single-molecule fluorescence imaging with single-particle tracking and localized and tracked single tRNA molecules over seconds. We observed two diffusive species: fast (with a diffusion coefficient of ∼8 μm2/s, consistent with free tRNA) and slow (consistent with tRNA bound to larger complexes). Our data indicate that a large fraction of internalized fluorescent tRNA (>70%) appears to diffuse freely in the bacterial cell. We also obtained the subcellular distribution of fast and slow diffusing tRNA molecules in multiple cells by normalizing for cell morphology. While fast diffusing tRNA is not excluded from the bacterial nucleoid, slow diffusing tRNA is localized to the cell periphery (showing a 30% enrichment versus a uniform distribution), similar to non-uniform localizations previously observed for mRNA and ribosomes.

Electrophoretic Deformation of Individual Transfer RNA Molecules Reveals Their Identity

It has been hypothesized that the ribosome gains additional fidelity during protein translation by probing structural differences in tRNA species. We measure the translocation kinetics of different tRNA species through ∼3 nm diameter synthetic nanopores. Each tRNA species varies in the time scale with which it is deformed from equilibrium, as in the translocation step of protein translation. Using machine-learning algorithms, we can differentiate among five tRNA species, analyze the ratios of tRNA binary mixtures, and distinguish tRNA isoacceptors.

Dicodon monitoring of protein synthesis (DiCoMPS) reveals levels of synthesis of a viral protein in single cells

The current report represents a further advancement of our previously reported technology termed Fluorescent transfer RNA (tRNA) for Translation Monitoring (FtTM), for monitoring of active global protein synthesis sites in single live cells. FtTM measures Förster resonance energy transfer (FRET) signals, generated when fluorescent tRNAs (fl-tRNAs), separately labeled as a FRET pair, occupy adjacent sites on the ribosome. The current technology, termed DiCodon Monitoring of Protein Synthesis (DiCoMPS), was developed for monitoring active synthesis of a specific protein. In DiCoMPS, specific fl-tRNA pair combinations are selected for transfection, based on the degree of enrichment of a dicodon sequence to which they bind in the mRNA of interest, relative to the background transcriptome of the cell in which the assay is performed. In this study, we used cells infected with the Epizootic Hemorrhagic Disease Virus 2-Ibaraki and measured, through DiCoMPS, the synthesis of the viral non-structural protein 3 (NS3), which is enriched in the AUA:AUA dicodon. fl-tRNAIleUAU-generated FRET signals were specifically enhanced in infected cells, increased in the course of infection and were diminished on siRNA-mediated knockdown of NS3. Our results establish an experimental approach for the single-cell measurement of the levels of synthesis of a specific viral protein.

Mechanism and dynamics of the elongation cycle

Continued dramatic progress in the elucidation of the structures of the bacterial ribosome and its functional complexes has led to proposals for the detailed mechanisms of ribosome-catalyzed protein synthesis (Schmeing and Ramakrishnan, 2009; Agirrezabala and Frank, 2009). Ensemble rapid reaction kinetics (Antoun et al., 2006; Daviter et al., 2006; Dorner et al., 2006; Grigoriadou et al., 2007; Hetricket al., 2009; Pan et al., 2007, 2008; Pape et al., 1998; Phelps and Joseph 2006; Rodnina et al., 1997; Savelsbergh et al., 2003; Walker et al., 2008; Wintermeyer et al., 2004; Zaher and Green, 2009; Zavialov and Ehrenberg, 2003) and single-molecule (Blanchard et al., 2004a, b; Cornish et al., 2008, 2009; Fei et al., 2008, 2009; Marshall et al., 2008, 2009; Munro et al., 2007, 2010a, b; Uemura et al., 2010; Wang et al., 2007) studies of the translational machinery in the past several years have resulted in increased understanding of many aspects of the initiation, elongation, and termination phases of protein synthesis, but many essential points remain to be elucidated.

Monitoring Collagen Synthesis in Fibroblasts Using Fluorescently Labeled tRNA Pairs

There is a critical need for techniques that directly monitor protein synthesis within cells isolated from normal and diseased tissue. Fibrotic disease, for which there is no drug treatment, is characterized by the overexpression of collagens. Here, we use a bioinformatics approach to identify a pair of glycine and proline isoacceptor tRNAs as being specific for the decoding of collagen mRNAs, leading to development of a FRET‐based approach, dicodon monitoring of protein synthesis (DiCoMPS), that directly monitors the synthesis of collagen. DiCoMPS aimed at detecting collagen synthesis will be helpful in identifying novel anti‐fibrotic compounds in cells derived from patients with fibrosis of any etiology, and, suitably adapted, should be widely applicable in monitoring the synthesis of other proteins in cells. J. Cell. Physiol. 229: 1121–1129, 2014.

Fluorescence enhancement on silver nanostructures: studies of components of ribosomal translation in vitro

Metallic particles, silver in particular, can significantly enhance the fluorescence of dye molecules in the immediate vicinity (5-20 nm) of the particle. This magnifying effect can be theoretically explained/predicted by considering the change of photonic mode density near the fluorophore due to coupling to the conducting surface. We are using this method to observe fluorescence from a single ribosomal particle in a project aimed at acquiring sequence information from the translating ribosome (NIHs

Single FV and VF events detected by FRET between Cy3-F and Cy5-V during translation of mRNA-1 (FV: A, B) and mRNA-2 (VF: C, D).

1000 Genome Initiative). Several quartz slides with silver nanostructures were made using electron beam lithography techniques. The structures were approximately 50 nm high silver tiles measuring 400-700 nm on the side, and were spaced differently over a total area of 1 mm x 1 mm on any given quartz slide. In a preliminary experiment, we coated this surface with the Alexa 647-labeled antibodies and collected single molecule images using the MicroTime 200 (PicoQuant) confocal system. We showed that the fluorescence intensity measured over the silver islands film was more than 100-fold higher than fluorescence from a comparable site on uncoated section of the quartz slide. No noticeable photobleaching was seen. The fluorescence lifetime was very short, about 200 ps or less (this is the resolution limit of the system). The method has great promise for investigations of biologically relevant single molecules.