In recent years, highly stable optical tweezers systems have enabled the characterization of the dynamics of molecular motors at very high resolution. However, the motion of many motors with angstrom-scale dynamics cannot be consistently resolved due to poor signal-to-noise ratio. Using an acousto-optic deflector to generate a "time-shared" dual-optical trap, we decreased low-frequency noise by more than one order of magnitude compared with conventional dual-trap optical tweezers. Using this instrument, we implemented a protocol that synthesizes single base-pair trajectories, which are used to test a Large State Space Hidden Markov Model algorithm to recover their individual steps. We then used this algorithm on real transcription data obtained in the same instrument to fully uncover the molecular trajectories of Escherichia coli RNA polymerase. We applied this procedure to reveal the effect of pyrophosphate on the distribution of dwell times between consecutive polymerase steps.

During protein synthesis, the ribosome translocates along a messenger RNA (mRNA) in one-codon steps catalyzed by the activity of a GTPase elongation factor, EF-G. Secondary structures in an mRNA pose a mechanical barrier to translocation as the mRNA entry pore on the ribosome only accommodates single-stranded RNA, and thus they must be opened prior to translocation. This opening is facilitated by several conserved positively charged amino acid side chains located at the entry pore. It is not clear, however, whether the ribosome disrupts the secondary structure at the mRNA entry site prior to translocation or concomitantly with translocation. Here, we measure both hairpin opening and EF-G binding to the ribosome using optical tweezers with single molecule fluorescence capability. We find that EF-G arrival precedes the opening of an mRNA hairpin and EF-G release occurs after the hairpin is opened, providing direct evidence that the unwinding action of the ribosome is concomitant with translocation. Furthermore, we occasionally observe an unwinding intermediate where the hairpin is opened in two successive half-codon steps while EF-G remains bound to the ribosome. Current models posit that translocation occurs through the reverse rotation of the small subunit head domain. Our results suggest that in the presence of a barrier, this reverse rotation might occur in two resolvable sub-steps. Finally, we find that while a strong mechanical barrier does not significantly decrease the EF-G dependent unwinding rate, it biases ribosomes into a kinetically altered, 10-fold slower pathway prior to translocation, resulting in a dramatic reduction of the global translation rate. We propose that such a slower pathway could be used to tune translation rate for nascent chain folding.

Programmed -1 ribosomal frameshifting(-1PRF) is tightly regulated by messenger RNA (mRNA) sequences and structures in expressing two or more proteins with precise ratios from a single mRNA. Using single-molecule fluorescence resonance energy transfer (smFRET) between (Cy5) EF-G and (Cy3)tRNA(Lys), we studied the translational elongation dynamics of -1PRF in the Escherichia coli dnaX gene, which contains three frameshifting signals: a slippery sequence (A AAA AAG), a Shine-Dalgarno (SD) sequence and a downstream hairpin. The frameshift promoting signals mostly impair the EF-G-catalyzed translocation step of the two tRNALys and the slippery codons from the A- and P- sites. The hairpin acts as a road block slowing the translocation rate. The upstream SD sequence together with the hairpin promotes dissociation of futile EF-G and thus causes multiple EF-G driven translocation attempts. A slippery sequence also helps dissociation of the EF-G by providing alternative base-pairing options. These results indicate that frameshifting takes place during the repetitive ribosomal conformational changes associated with EF-G dissociation upon unsuccessful translocation attempts of the second slippage codon from the A- to the P- sites.

Protein synthesis rates can affect gene expression and the folding and activity of the translation product. Interactions between the nascent polypeptide and the ribosome exit tunnel represent one mode of regulating synthesis rates. The SecM protein arrests its own translation, and release of arrest at the translocon has been proposed to occur by mechanical force. Using optical tweezers, we demonstrate that arrest of SecM-stalled ribosomes can indeed be rescued by force alone and that the force needed to release stalling can be generated in vivo by a nascent chain folding near the ribosome tunnel exit. We formulate a kinetic model describing how a protein can regulate its own synthesis by the force generated during folding, tuning ribosome activity to structure acquisition by a nascent polypeptide.

Programmed ribosomal frameshifting produces alternative proteins from a single transcript. -1 frameshifting occurs on Escherichia coli's dnaX mRNA containing a slippery sequence AAAAAAG and peripheral mRNA structural barriers. Here, we reveal hidden aspects of the frameshifting process, including its exact location on the mRNA and its timing within the translation cycle. Mass spectrometry of translated products shows that ribosomes enter the -1 frame from not one specific codon but various codons along the slippery sequence and slip by not just -1 but also -4 or +2 nucleotides. Single-ribosome translation trajectories detect distinctive codon-scale fluctuations in ribosome-mRNA displacement across the slippery sequence, representing multiple ribosomal translocation attempts during frameshifting. Flanking mRNA structural barriers mechanically stimulate the ribosome to undergo back-and-forth translocation excursions, broadly exploring reading frames. Both experiments reveal aborted translation around mutant slippery sequences, indicating that subsequent fidelity checks on newly adopted codon position base pairings lead to either resumed translation or early termination.

My personal view of ethical behavior as a scientific researcher in an academic environment is presented. I discuss the behavior of a graduate student, a postdoctoral, and a professor. Ethical behavior in teaching, choosing a research project, publishing papers, and obtaining a job is discussed. © 2014 Wiley Periodicals, Inc. Biopolymers 103: 424–431, 2015.

Translational frameshifting occurs when a ribosome slips one or two nucleotides on a messenger RNA and generates a new sequence of amino acids. Many viral RNAs have programed frameshift-promoting signals to produce their proteins in the precise ratio needed for their viability. We used single-molecule fluorescence resonance energy transfer (smFRET) to study the dynamics of −1 programmed frameshifting by the dnaX gene in E. coli. The frameshifting mRNA has the usual three prokaryotic frameshifting signals: an internal Shine-Dalgarno sequence, a slippery sequence, and a stem loop. One round of translational elongation of the slippery sequence was characterized by the FRET changes between a Cy3-labeled L1 stalk in the 50S subunit and a Cy5-tRNALys in the P-site. We observed that the downstream stem loop, a critical signal for efficient frameshifting, destabilizes the hybrid state and thus shifts the equilibrium toward to the classical state of pre-translocation complexes. Translocation catalyzed by EF-G was significantly slower in the frameshifting mRNA than in the non-frameshifting mRNA lacking the stem loop. Furthermore, pre-translocation complexes of the frameshifting mRNA underwent several transitions between the classical and hybrid states in the presence of EF-G prior to complete translocation, while the majority of the non-frameshifting mRNA translocated rapidly via a single hybrid state. Quantitative analysis showed that the stem loop impedes EF-G driven translocation in the 30S subunit by elevating the activation barriers to translocation, and leaves the EF-G bound-hybrid state in dynamic equilibrium with the hybrid and classical states. We propose that by keeping the ribosome and tRNAs in the dynamically transiting pre-translocation states, the frameshifting mRNA allows more time for the ribosome to explore other paths, such as −1 frameshifting.

Life at Berkeley for the past 57 years involved research on the thermodynamics, kinetics, and spectroscopic properties of RNA to better understand its structures, interactions, and functions. We (myself and all the graduate students and postdocs who shared in the fun) began with dinucleoside phosphates and slowly worked our way up to megadalton-sized RNA molecular motors. We used UV absorption, circular dichroism, circular intensity differential scattering, fluorescence, NMR, and single-molecule methods. We learned a lot and had fun doing it.

Ribosomes programmed by specific messenger RNA (mRNA) sequence elements can switch translation reading frames and synthesize different polypeptides from a single template. The Escherichia coli dnaX mRNA encodes two DNA polymerase III subunits, τ and γ, synthesized from 0-frame and probabilistic −1-slip across the slippery sequence: AAAAAAG. When further enhanced by structural barriers situated around the slippery sequence-an internal Shine-Dalgarno sequence and a stable hairpin stem loop, an 80% (= γ/(γ+τ)) frameshift efficiency is attained. Here, we attempt to determine the frameshift timing within one translation cycle by following a single ribosome translating a frameshift-promoting mRNA held on optical tweezers. In parallel, by mass spectrometry (MS), we survey the synthesized polypeptides to resolve where on the mRNA the ribosome has slipped. From the mass spectra of polypeptides terminated at the −1-frame stop codon, we learned that the ribosome −1-slips from more than one codon position around the slippery sequence. Some −1-frameshifted polypeptides were found to bear an extra amino acid, or to lack one, indicating that slipping sizes of −4 and +2-nt also occurred. Similarly, distinctive large-scale fluctuating translocation dynamics were seen in our real-time single-ribosome translation trajectories. This reveals that a translocating ribosome can explore a broad range of frameshift pathways. Frequently adopted frameshift pathways, i.e. the more abundant frameshifted species resolved by MS, exhibit a preference for minimizing codon:anticodon base-pair mismatches on the ribosome after a slip. Mismatch-containing ribosomes can be prematurely terminated by release factors, resulting in release of incomplete peptides. Indeed, we observed higher yields of incomplete peptides that are terminated at frameshift sites where significant mismatches were encountered. These species coincide with the prematurely stalled ribosomes recorded in the translation trajectories. Collectively what emerges from our results is a versatile ribosomal frameshifting scheme during mRNA translocation, facilitating branching of frameshift pathways.

A detailed understanding of tRNA/mRNA translocation requires measurement of the forces generated by the ribosome during this movement. Such measurements have so far remained elusive and, thus, little is known about the relation between force and translocation and how this reflects on its mechanism and regulation. Here, we address these questions using optical tweezers to follow translation by individual ribosomes along single mRNA molecules, against an applied force. We find that translocation rates depend exponentially on the force, with a characteristic distance close to the one-codon step, ruling out the existence of sub-steps and showing that the ribosome likely functions as a Brownian ratchet. We show that the ribosome generates ∼13 pN of force, barely sufficient to unwind the most stable structures in mRNAs, thus providing a basis for their regulatory role. Our assay opens the way to characterizing the ribosome's full mechano–chemical cycle.

Programmed frameshifting is used by prokaryotes and eukaryotes to synthesize two or more proteins from the same messenger RNA. We have studied minus‐one frameshifting in the dnaX gene in E. coli, whose mRNA contains the usual frameshifting signals: an internal Shine Dalgarno sequence, a slippery sequence (AAAAAAG), and a stem‐loop. We used bulk mass spectrometry, single‐molecule laser tweezers, and single‐molecule FRET in our studies. We found that minus‐one frameshifting occurs at the Lys codons in the slippery sequence, but also at codons on either side. Furthermore, the minus‐one frameshift occurs by slips of the ribosome of ‐1, +2, or even ‐4 nucleotides. The translation trajectories show step‐by‐step progression as each codon is translated, but at the slippery sequence large‐scale fluctuations in position of the ribosome are seen. This agrees with the mass spectrometry results showing multiple sites and multiple paths of frameshifting.After peptide bond formation the tRNAs undergo a classic‐hybrid equilibrium before the elongation factor, EF‐G•GTP, catalyzes the translocation. Our single‐molecule FRET studies of fluorophore‐labeled ribosome and tRNA found that the presence of the stem‐loop stabilizes the ribosome in the hybrid state before translocation. The longer time spent in this state allows the ribosome to sample other states, and thus may favor sites and paths in addition to a zero‐frame move.

In the article entitled, “Frameshifting Dynamics,” Biopolymers 2013;99(12):1147–1166, a minor error in Figure 3 was published. The authors apologize for the error and any inconvenience caused.

ADVERTISEMENT RETURN TO ISSUEPREVReviewNEXTProbing the Mechanisms of Translation with ForceChristian M. Kaiser† and Ignacio Tinoco, Jr.*‡View Author Information‡ †QB3 Institute, and ‡Department of Chemistry, University of California, Berkeley, Berkeley, California 94720, United States*E-mail: [email protected]Cite this: Chem. Rev. 2014, 114, 6, 3266–3280Publication Date (Web):January 9, 2014Publication History Received10 June 2013Published online9 January 2014Published inissue 26 March 2014https://pubs.acs.org/doi/10.1021/cr400313xhttps://doi.org/10.1021/cr400313xreview-articleACS PublicationsCopyright © 2014 American Chemical SocietyRequest reuse permissionsArticle Views1397Altmetric-Citations14LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-Alertsclose SUBJECTS:Deformation,Free energy,Genetics,Monomers,Peptides and proteins Get e-Alerts

Significance A ribosomal frameshift occurs when the ribosome slips by one or more nucleotides on the messenger RNA (mRNA) during translation. Programmed ribosomal frameshifting produces more than one protein from a single mRNA and is tightly regulated by mRNA sequence and structure. Using single-molecule fluorescence resonance energy transfer, we studied the effects of a frameshifting stimulatory mRNA structure on the ribosomal conformational dynamics and translocation rate. Our results show that the structure shifts the conformational equilibrium of ribosomal complexes away from conformations that favor the translocation process, resulting in slowed translocation. We propose that the downstream structure traps ribosomal complexes in the fluctuating conformational states of the translocation process and thus allows more opportunities for frameshifting.

Translocation of tRNA and mRNA by the ribosome during protein synthesis involves a number of precise and coordinated macromolecular rearrangements. Recently, high-resolution x-ray structures, cryo-EM reconstructions, and single-molecule fluorescence studies have yielded insight into the nature of these conformational states, and the kinetics of their inter-conversion. Yet, a detailed understanding of translocation is still missing mainly because its characterization requires the application of external force to inhibit or facilitate the step in which the ribosome converts chemical energy into mechanical work. Such measurements have so far remained elusive and, as a result, little is known about the mechanical properties of the ribosome, the maximum force it can exert during translocation, and its thermodynamic efficiency. Moreover, it is not known how the application of an external force affects the translocation rate, a response that is relevant to understanding the mechanism of translocation, co-translational protein folding, frameshifting, and other forms of translation regulation. Here, we address these questions using optical tweezers to follow translation by individual ribosomes along single mRNA molecules, against externally applied force. We find that the transition state during translocation is located close to the full extent of a one-codon step, ruling out models in which the mechanical step is composed of some combination of one- or two-nucleotide sub-steps. We show that the ribosome is able to generate a force barely sufficient to unwind the most stable structures typically found in mRNAs, making the translation rate highly sensitive to the presence and stability of these structures, and providing a mechanical basis for their regulatory role. Finally, our measurements indicate that the ribosome is able to convert the energy from the transpeptidation reaction into mechanical work with high efficiency. Our assay opens the way to characterizing the full mechano-chemical cycle of the ribosome.

Translation of messenger RNA by a ribosome occurs three nucleotides at a time from start signal to stop. However, a frameshift means that some nucleotides are read twice or some are skipped, and the following sequence of amino acids is completely different from the sequence in the original frame. In some messenger RNAs, including viral RNAs, frameshifting is programmed with RNA signals to produce specific ratios of proteins vital to the replication of the organism. The mechanisms that cause frameshifting have been studied for many years, but there are no definitive conclusions. We review ribosome structure and dynamics in relation to frameshifting dynamics provided by classical ensemble studies, and by new single-molecule methods using optical tweezers and FRET.

1321-Pos Board B213 Co-Translational Protein Folding on the Ribosome: using NMR Spectroscopy to Provide Structure and Dynamics of Ribosome-Nascent Chains John Christodoulou. University College London, London, United Kingdom. The folding processes of nascent chains are intricately linked to their chain elongation, which occurs in a vectorial manner as the N-terminal part of the nascent chain emerges from the ribosome [1]. The use of NMR spectroscopy on ribosomes and ribosome nascent-chain complexes (RNCs) is providing detailed structural insights of the conformations of protein chains while they are being created on the ribosome. By producing in-vivo derived RNCs in which the nascent polypeptide is selectively labelled, our recent work has allowed us to use NMR to follow, at a residue-specific level, the co-translational folding processes of proteins of several topologies, specifically, an immunoglobulin (Ig) domain, of YFP (alongside fluorescence) and of the intrinsically disordered protein, alpha-synuclein. New work to be described here is allowing us to describe the types of intermediates structures sampled during the vectorial emergence, the interactions of the emerging chains with the ribosome and also how the molecular chaperone, the trigger factor, that interacts with the nascent chain, affect protein folding. Recent strides towards a detailed understanding of the relationship between biosynthesis and folding will be discussed. Past references: 1. Protein Folding on theRibosomeCabrita, L.D., Dobson,C.M., Christodoulou, J.* Current Opin Struct Biol (2010) 20, 33-45. 2. Probing ribosome-nascent chain complexes produced in vivo by NMR spectroscopy Cabrita, L.D., Hsu, S-T. D., Launay, H., Dobson, C.M., Christodoulou, J.* P.N.A.S (2009) 106, 22239-44 3. New scenarios of protein folding can occur on the ribosome O’Brien, E.P., Christodoulou, J., Vendruscolo, M., Dobson, C.M. J. Am. Chem. Soc (2011) 133, 513–526 4. Tertiary structure formation inside the ribosome exit port O’Brien, E.P., Hsu, S-T. D., Christodoulou, J., Vendruscolo, M., Dobson, C.M. J. Am. Chem. Soc (2010) 32, 16928-37.

We have investigated the regulation of fibronectin and procollagen synthesis in normal and Rous sarcoma virus transformed primary avian tendon cells These two proteins interact at the cell periphery and both are reportedly lost upon transformation. We thus examined whether their synthesis was coordinately regulated in RSV-infected cells. It was found that while the synthesis of both pro ctl and pro a2 peptides was reduced upon transformation, the synthesis of fibronectin was not altered Nevertheless, lông term radiolabeling demonstrated that fibronectin levels were reduced in transformed cells. It is concluded that the reduction in levels of these components at the surface is brought about by different mechanisms, collagen levels being regulated by procollagen synthesis and fibronectin levels • by degradation and/or release into the culture medium. The possibility is discussed that fibronectin is lost from the cell periphery of PAT cells as a consequence of decreased levels of anChoring collagen mo.leules

The sequence and secondary structure of the 5′-end of mRNAs regulate translation by controlling ribosome initiation on the mRNA. Ribosomal protein S1 is crucial for ribosome initiation on many natural mRNAs, particularly for those with structured 5′-ends, or with no or weak Shine-Dalgarno sequences. Besides a critical role in translation, S1 has been implicated in several other cellular processes, such as transcription recycling, and the rescuing of stalled ribosomes by tmRNA. The mechanisms of S1 functions are still elusive but have been widely considered to be linked to the affinity of S1 for single-stranded RNA and its corresponding destabilization of mRNA secondary structures. Here, using optical tweezers techniques, we demonstrate that S1 promotes RNA unwinding by binding to the single-stranded RNA formed transiently during the thermal breathing of the RNA base pairs and that S1 dissociation results in RNA rezipping. We measured the dependence of the RNA unwinding and rezipping rates on S1 concentration, and the force applied to the ends of the RNA. We found that each S1 binds 10 nucleotides of RNA in a multistep fashion implying that S1 can facilitate ribosome initiation on structured mRNA by first binding to the single strand next to an RNA duplex structure (“stand-by site”) before subsequent binding leads to RNA unwinding. Unwinding by multiple small substeps is much less rate limited by thermal breathing than unwinding in a single step. Thus, a multistep scheme greatly expedites S1 unwinding of an RNA structure compared to a single-step mode.