Benjamin H. Stevens

Single-Molecule Fluorescence Measurements of Ribosomal Translocation Dynamics

We employ single-molecule fluorescence resonance energy transfer (smFRET) to study structural dynamics over the first two elongation cycles of protein synthesis, using ribosomes containing either Cy3-labeled ribosomal protein L11 and A- or P-site Cy5-labeled tRNA or Cy3- and Cy5-labeled tRNAs. Pretranslocation (PRE) complexes demonstrate fluctuations between classical and hybrid forms, with concerted motions of tRNAs away from L11 and from each other when classical complex converts to hybrid complex. EF-G⋅GTP binding to both hybrid and classical PRE complexes halts these fluctuations prior to catalyzing translocation to form the posttranslocation (POST) complex. EF-G dependent translocation from the classical PRE complex proceeds via transient formation of a short-lived hybrid intermediate. A-site binding of either EF-G to the PRE complex or of aminoacyl-tRNA⋅EF-Tu ternary complex to the POST complex markedly suppresses ribosome conformational lability.

Allosteric vs. spontaneous exit-site (E-site) tRNA dissociation early in protein synthesis

During protein synthesis, deacylated transfer RNAs leave the ribosome via an exit (E) site after mRNA translocation. How the ribosome regulates tRNA dissociation and whether functional linkages between the aminoacyl (A) and E sites modulate the dynamics of protein synthesis have long been debated. Using single molecule fluorescence resonance energy transfer experiments, we find that, during early cycles of protein elongation, tRNAs are often held in the E site until being allosterically released when the next aminoacyl tRNA binds to the A site. This process is regulated by the length and sequence of the nascent peptide and by the conformational state, detected by tRNA proximity, prior to translocation. In later cycles, E-site tRNA dissociates spontaneously. Our results suggest that the distribution of pretranslocation tRNA states and posttranslocation pathways are correlated within each elongation cycle via communication between distant subdomains in the ribosome, but that this correlation between elongation cycle intermediates does not persist into succeeding cycles.

Enhancement of Single Molecule Fluorescence Signals by Colloidal Silver Nanoparticles in Studies of Protein Translation

Metal-enhanced fluorescence (MEF) increased total photon emission of Cy3- and Cy5-labeled ribosomal initiation complexes near 50 nm silver particles 4- and 5.5-fold, respectively. Fluorescence intensity fluctuations above shot noise, at 0.1-5 Hz, were greater on silver particles. Overall signal-to-noise ratio was similar or slightly improved near the particles. Proximity to silver particles did not compromise ribosome function, as measured by codon-dependent binding of fluorescent tRNA, dynamics of fluorescence resonance energy transfer between adjacent tRNAs in the ribosome, and tRNA translocation induced by elongation factor G.

A Study of the Role of Regionalization in the Generation of Aggregation Error in Regional Input –Output Models

Although the need for aggregation in input -output modelling has diminished with the increases in computing power, an alarming number of regional studies continue to use the procedure. The rationales for doing so typically are grounded in data problems at the regional level. As a result many regional analysts use aggregated national input -output models and trade -adjust them at this aggregated level. In this paper, we point out why this approach can be inappropriate. We do so by noting that it creates a possible source of model misapplication (i.e., a direct effect could appear for a sector where one does not exist) and also by finding that a large amount of error (on the order of 100 percent) can be induced into the impact results as a result of improper aggregation. In simulations, we find that average aggregation error tends to peak at 81 sectors after rising from 492 to 365 sectors. Perversely, error then diminishes somewhat as the model size decreases further to 11 and 6 sectors. We also find that while region - and sector -specific attributes influence aggregation error in a statistically significantly manner, their influence on the amount of error generally does not appear to be large. Copyright 2002 Blackwell Publishers Inc.

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.

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.