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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.

Force to unblock a clogged ribosome The synthesis of proteins from mRNA by the ribosome is highly regulated. But newly synthesized protein chains can still block the ribosome exit tunnel and slow protein synthesis. Goldman et al. use optical tweezers to show that by pulling on the stuck protein chain, they can unblock a clogged exit tunnel (see the Perspective by Puglisi). In vivo, the folding of a nascent protein chain just outside the tunnel also generates enough force to unclog a block, indicating that ribosome-peptide interactions fine-tune protein synthesis. Science , this issue p. 457; see also p. 399

Significance Optical tweezers enable scientists to follow the dynamics of molecular motors at high resolution. The ability to discern a motor’s discrete steps reveals important insights on its operation. Some motors operate at the scale of angstroms, rendering the observation of their steps extremely challenging. In some cases, such small steps have been observed sporadically; however, the full molecular trajectories of steps and intervals between steps remain elusive due to instrumental noise. Here, we eliminate the main source of noise of most high-resolution dual-trap optical tweezers and developed both a single-molecule assay and a self-learning algorithm to uncover the full trajectories of such a motor: RNA polymerase. Using this method, a whole new set of experiments becomes possible., 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.

Summary 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.

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.

The movement of ribosomes on mRNA is often interrupted by secondary structures that present mechanical barriers and play a central role in translation regulation. We investigate how ribosomes couple their internal conformational changes with the activity of translocation factor EF-G to unwind mRNA secondary structures using high-resolution optical tweezers with single-molecule fluorescence capability. We find that hairpin opening occurs during EF-G-catalyzed translocation and is driven by the forward rotation of the small subunit head. Modulating the magnitude of the hairpin barrier by force shows that ribosomes respond to strong barriers by shifting their operation to an alternative 7-fold-slower kinetic pathway prior to translocation. Shifting into a slow gear results from an allosteric switch in the ribosome that may allow it to exploit thermal fluctuations to overcome mechanical barriers. Finally, we observe that ribosomes occasionally open the hairpin in two successive sub-codon steps, revealing a previously unobserved translocation intermediate.

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)tRNALys, 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.

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.

Nonhexameric helicases use adenosine triphosphate (ATP) to unzip base pairs in double-stranded nucleic acids (dsNAs). Studies have suggested that these helicases unzip dsNAs in single–base pair increments, consuming one ATP molecule per base pair, but direct evidence for this mechanism is lacking. We used optical tweezers to follow the unwinding of double-stranded RNA by the hepatitis C virus NS3 helicase. Single–base pair steps by NS3 were observed, along with nascent nucleotide release that was asynchronous with base pair opening. Asynchronous release of nascent nucleotides rationalizes various observations of its dsNA unwinding and may be used to coordinate the translocation speed of NS3 along the RNA during viral replication.

An RNA kissing complex formed by the dimerization initiation site plays a critical role in the survival and infectivity of human immunodeficiency virus. Two dimerization initiation site kissing sequences, Mal and Lai, have been found in most human immunodeficiency virus 1 variants. Formation and stability of these RNA kissing complexes depend crucially on cationic conditions, particularly Mg 2+. Using optical tweezers, we investigated the mechanical unfolding of single RNA molecules with either Mal-type (GUGCAC) or Lai-type (GCGCGC) kissing complexes under various ionic conditions. The force required to disrupt the kissing interaction of the two structures, the rip force, is sensitive to concentrations of KCl and MgCl2; addition of 3 mM MgCl2 to 100 mM KCl changes the rip force of Mal from 21 +/- 4 to 46 +/- 3 pN. From the rip force distribution, the kinetics of breaking the kissing interaction is calculated as a function of force and cation concentration. The two kissing complexes have distinct unfolding transition states, as shown by different values of deltaX(++), which is the distance from the folded structure to the unfolding transition state. The deltaX(++) of Mal is approximately 0.6 nm smaller than that of Lai, suggesting that fewer kissing base pairs are broken at the transition state of the former, consistent with observations that the Lai-type kissing complex is more stable and requires significantly more force to unfold than the Mal type. More importantly, neither K+ nor Mg 2+ significantly changes the position of the transition state along the reaction coordinate. However, increasing concentrations of cations increase the kinetic barrier. We derived a cation-specific parameter, m, to describe how the height of the kinetic barrier depends on the concentration of cations. Our results suggest that Mg 2+ greatly slows down the unfolding of the kissing complex but has moderate effects on the formation kinetics of the structure.

Using optical tweezers, we have measured the effect of monovalent cation concentration and species on the folding free energy of five large (49-124 nt) RNA hairpins, including HIV-1 TAR and molecules approximating A.U and G.C homopolymers. RNA secondary structure thermodynamics are accurately described by a model consisting of nearest-neighbor interactions and additive loop and bulge terms. Melting of small (15 bp) duplexes and hairpins in 1 M NaCl has been used to determine the parameters of this model, which is now used extensively to predict structure and folding dynamics. Few systematic measurements have been made in other ionic conditions or for larger structures. By applying mechanical force, we measured the work required to fold and unfold single hairpins at room temperature over a range of cation concentrations from 50 to 1000 mM. Free energies were then determined using the Crooks fluctuation theorem. We observed the following: (1) In most cases, the nearest-neighbor model accurately predicted the free energy of folding at 1 M NaCl. (2) Free energy was proportional to the logarithm of salt concentration. (3) Substituting potassium ions for sodium slightly decreased hairpin stability. The TAR hairpin also misfolded nearly twice as often in KCl, indicating a differential kinetic response. (4) Monovalent cation concentration affects RNA stability in a sequence-dependent manner. G.C helices were unaffected by changing salt concentration, A.U helices were modestly affected, and the hairpin loop was very sensitive. Surprisingly, the U.C.U bulge of TAR was found to be equally stable in all conditions tested. We also report a new estimate for the elastic parameters of single-stranded RNA.

By exerting mechanical force it is possible to unfold/refold RNA molecules one at a time. In a small range of forces, an RNA molecule can hop between the folded and the unfolded state with force-dependent kinetic rates. Here, we introduce a mesoscopic model to analyze the hopping kinetics of RNA hairpins in an optical tweezers setup. The model includes different elements of the experimental setup (beads, handles and RNA sequence) and limitations of the instrument (time lag of the force-feedback mechanism and finite bandwidth of data acquisition). We investigated the influence of the instrument on the measured hopping rates. Results from the model are in good agreement with the experiments reported in the companion article (1). The comparison between theory and experiments allowed us to infer the values of the intrinsic molecular rates of the RNA hairpin alone and to search for the optimal experimental conditions to do the measurements. We conclude that long handles and soft laser traps represent the best conditions to extract rate estimates that are closest to the intrinsic molecular rates. The methodology and rationale presented here can be applied to other experimental setups and other molecules., Comment: PDF file, 32 pages including 9 figures plus supplementary material

1. Introduction 3262. Instrumentation 3282.1 Instruments to study mechanical properties of RNA 3282.1.1 AFM 3282.1.2 Magnetic tweezers 3282.1.3 Optical tweezers 3302.2 Optical trap instrumentation 3302.3 Calibrations 3322.3.1 Calibration of trap stiffness 3322.3.2 Calibration of force 3332.3.3 Calibration of distance 3342.4 Types of experiments 3342.4.1 Force-ramp 3342.4.2 Force-clamp or constant-force experiments 3352.4.3 Extension-clamp or constant extension experiments 3352.4.4 Force-jump, Force-drop 3362.4.5 Passive mode 3363. Thermodynamics 3363.1 Reversibility 3363.2 Gibbs free energy 3373.2.1 Stretching free energy 3383.2.1.1 Rigid molecules 3383.2.1.2 Compliant or flexible molecules 3393.2.2 Free energy of a reversible unfolding transition 3393.2.3 Free energy of unfolding at zero force 3403.2.4 Free energy of an irreversible unfolding transition 3403.2.4.1 Jarzynski's method 3413.2.4.2 Crooks fluctuation theorem 3434. Kinetics 3454.1 Measuring rate constants 3454.1.1 Hopping 3454.1.2 Force-jump, Force-drop 3474.1.3 Force-ramp 3484.1.4 Instrumental effects 3504.2 Kinetic mechanisms 3514.2.1 Free-energy landscapes 3514.2.2 Kinetics of unfolding 3535. Relating force-measured data to other measurements 3545.1 Thermodynamics 3545.2 Kinetics 3576. Acknowledgements 3577. References 358Single-molecule methods have made it possible to apply force to an individual RNA molecule. Two beads are attached to the RNA; one is on a micropipette, the other is in a laser trap. The force on the RNA and the distance between the beads are measured. Force can change the equilibrium and the rate of any reaction in which the product has a different extension from the reactant. This review describes use of laser tweezers to measure thermodynamics and kinetics of unfolding/refolding RNA. For a reversible reaction the work directly provides the free energy; for irreversible reactions the free energy is obtained from the distribution of work values. The rate constants for the folding and unfolding reactions can be measured by several methods. The effect of pulling rate on the distribution of force-unfolding values leads to rate constants for unfolding. Hopping of the RNA between folded and unfolded states at constant force provides both unfolding and folding rates. Force-jumps and force-drops, similar to the temperature jump method, provide direct measurement of reaction rates over a wide range of forces. The advantages of applying force and using single-molecule methods are discussed. These methods, for example, allow reactions to be studied in non-denaturing solvents at physiological temperatures; they also simplify analysis of kinetic mechanisms because only one intermediate at a time is present. Unfolding of RNA in biological cells by helicases, or ribosomes, has similarities to unfolding by force.

Mechanical unfolding and refolding of single RNA molecules have previously been observed in optical traps as sudden changes in molecular extension. Two methods have been traditionally used: “force-ramp”, with the applied force continuously changing, and “hopping”. In hopping experiments the force is held constant and the molecule jumps spontaneously between two different states. Unfolding/refolding rates are measured directly, but only over a very narrow range of forces. We have now developed a force-jump method to measure the unfolding and refolding rates independently over a wider range of forces. In this method, the applied force is rapidly stepped to a new value and either the unfolding or refolding event is monitored through changes in the molecular extension. The force-jump technique is compared to the force-ramp and hopping methods by using a 52-nucleotide RNA hairpin with a three-nucleotide bulge, i.e., the transactivation response region RNA from the human immunodeficiency virus. We find the unfolding kinetics and Gibbs free energies obtained from all three methods to be in good agreement. The transactivation response region RNA hairpin unfolds in an all-or-none two-state reaction at any loading rate with the force-ramp method. The unfolding reaction is reversible at small loading rates, but shows hysteresis at higher loading rates. Although the RNA unfolds and refolds without detectable intermediates in constant-force conditions (hopping and force-jump), it shows partially folded intermediates in force-ramp experiments at higher unloading rates. Thus, we find that folding of RNA hairpins can be more complex than a simple single-step reaction, and that application of several methods can improve understanding of reaction mechanisms.

Protein folding is often studied in the context of full-length polypeptides in solution. However, the post-translational folding pathway may not recreate the pathway of folding when a protein is being synthesized by the ribosome. The effects of the ribosome and the rate of translation on folding are not well understood, partially due to the difficulty of probing the nascent chain without also affecting the ribosome. Using optical tweezers, we can look in detail at the nascent chain at the single-molecule level with minimal perturbation to the ribosome. Previous studies have shown that the ribosomal surface can act electrostatically to slow the kinetics of folding. Those studies used a linker to vary the distance from the surface and did not look at changes in sequence availability or multidomain proteins. To further understand cotranslational folding, we are studying the folding pathway of a two-domain calcium-binding protein calerythrin. Our results show that the full-length protein in solution folds robustly through a C-domain intermediate, but truncated versions of the protein can fold to an N domain or a misfolded state. During translation, the C-terminal residues required for native folding are not yet available, but the other observed intermediates could fold, in principle. By observing the folding at various sequence positions, and thus nascent chain lengths, we found that the ribosome can not only slow folding, but can also affect the unfolding rates, particularly increasing the unfolding rate of the misfolded state. The ribosome thus prevents the formation of this unproductive state via two mechanisms: decreasing the probability of folding and decreasing the stability of the folded state. This study gives further insight into the importance of the ribosome to regulate protein structure, and opens up new questions about the interplay between elongation and folding.

Mechanical unfolding trajectories for single molecules of the Tetrahymena thermophila ribozyme display eight intermediates corresponding to discrete kinetic barriers that oppose mechanical unfolding with lifetimes of seconds and rupture forces between 10 and 30 piconewtons. Barriers are magnesium dependent and correspond to known intra- and interdomain interactions. Several barrier structures are “brittle,” breakage requiring high forces but small (1 to 3 nanometers) deformations. Barrier crossing is stochastic, leading to variable unfolding paths. The response of complex RNA structures to locally applied mechanical forces may be analogous to the responses of RNA during translation, messenger RNA export from the nucleus, and viral replication.

The usual variables chemists use to affect a chemical reaction are temperature and pressure. We consider here an additional variable: force, F. By attaching a molecule to the tip of a cantilever of an atomic force microscope, or to a bead in a laser light trap, we can control the force on a single molecule. This mechanical force can drive a reaction to completion, or stabilize the reactants. Force changes the thermodynamic stability of a molecule; it can thus increase or decrease the free energy change for the reaction. Force can also speed or slow rates of reactions; it changes the free energy of activation of the reaction.

We investigate the work dissipated during the irreversible unfolding of single molecules by mechanical force, using the simplest model necessary to represent experimental data. The model consists of two levels (folded and unfolded states) separated by an intermediate barrier. We compute the probability distribution for the dissipated work and give analytical expressions for the average and variance of the distribution. To first order, the amount of dissipated work is directly proportional to the rate of application of force (the loading rate), and to the relaxation time of the molecule. The model yields estimates for parameters that characterize the unfolding kinetics under force in agreement with those obtained in recent experimental results (Liphardt, J., et al. (2002) {\em Science}, {\bf 296} 1832-1835). We obtain a general equation for the minimum number of repeated experiments needed to obtain an equilibrium free energy, to within $k_BT$, from non-equilibrium experiments using the Jarzynski formula. The number of irreversible experiments grows exponentially with the ratio of the average dissipated work, $\bar{\Wdis}$, to $k_BT$.}, Comment: PDF file, 5 pages

Recent advances in statistical mechanical theory can be used to solve a fundamental problem in experimental thermodynamics. In 1997, Jarzynski proved an equality relating the irreversible work to the equilibrium free energy difference, Δ G . This remarkable theoretical result states that it is possible to obtain equilibrium thermodynamic parameters from processes carried out arbitrarily far from equilibrium. We test Jarzynski's equality by mechanically stretching a single molecule of RNA reversibly and irreversibly between two conformations. Application of this equality to the irreversible work trajectories recovers the Δ G profile of the stretching process to within k B T /2 (half the thermal energy) of its best independent estimate, the mean work of reversible stretching. The implementation and test of Jarzynski's equality provides the first example of its use as a bridge between the statistical mechanics of equilibrium and nonequilibrium systems. This work also extends the thermodynamic analysis of single molecule manipulation data beyond the context of equilibrium experiments.

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. DOI: http://dx.doi.org/10.7554/eLife.03406.001, eLife digest Producing a protein first requires its gene to be transcribed into a long molecule called a messenger RNA (mRNA). A complex molecular machine called the ribosome then translates the mRNA code by reading it three letters at a time. Each triplet of letters—known as a codon—tells the ribosome which amino acid to add next into the protein. After adding an amino acid, the ribosome moves along the mRNA molecule to read the next codon and add another amino acid into the protein chain. While researchers understand how protein chains are formed, how the ribosome shifts along the mRNA strand—a process called translocation—is still unclear. It is known that this process involves many force-generating movements and changes to the shape of the ribosome. However, it is only recently that researchers have been able to measure these forces. Using optical tweezers—an instrument that uses a highly focused laser beam to hold and manipulate microscopic objects—Liu, Kaplan et al. followed individual ribosomes as they translated an mRNA and measured the effect that applying an opposing force has on the rate of translation. The results shed new light on the mechanism of translocation. First, Liu, Kaplan et al. found that ribosomes jump directly from one triplet to the next in the mRNA sequence, rather than moving there in a series of smaller steps. Next, the results indicate that translocation occurs spontaneously, driven by thermal energy, while chemical reactions prevent the reverse movement, in a mechanism known as a ‘Brownian Ratchet’. Measurements of the maximum force generated by the ribosome also give insights into how translation is regulated. Strands of mRNA can fold into certain structures that slow down translation, because the mRNA must first be unfolded before the ribosome can translate it. Liu, Kaplan et al. found that the maximum force generated by a ribosome is only just enough to unwind these mRNA structures, making the translation rate highly sensitive to the existence of such structures, and the structures themselves of high importance for regulating transcription. Given its importance as the ultimate decoder of the genetic information, understanding the ribosome's function and regulation has broad implications. The work of Liu, Kaplan et al. opens the way for a full characterization of the role of mechanical forces in the translation process. DOI: http://dx.doi.org/10.7554/eLife.03406.002

Ribosomal frameshifting occurs when a ribosome slips a few nucleotides on an mRNA and generates a new sequence of amino acids. Programmed -1 ribosomal frameshifting (-1PRF) is used in various systems to express two or more proteins from a single mRNA at precisely regulated levels. We used single-molecule fluorescence resonance energy transfer (smFRET) to study the dynamics of -1PRF in the Escherichia coli dnaX gene. The frameshifting mRNA (FSmRNA) contained the frameshifting signals: a Shine-Dalgarno sequence, a slippery sequence, and a downstream stem loop. The dynamics of ribosomal complexes translating through the slippery sequence were characterized using smFRET between the Cy3-labeled L1 stalk of the large ribosomal subunit and a Cy5-labeled tRNA(Lys) in the ribosomal peptidyl-tRNA-binding (P) site. We observed significantly slower elongation factor G (EF-G)-catalyzed translocation through the slippery sequence of FSmRNA in comparison with an mRNA lacking the stem loop, ΔSL. Furthermore, the P-site tRNA/L1 stalk of FSmRNA-programmed pretranslocation (PRE) ribosomal complexes exhibited multiple fluctuations between the classical/open and hybrid/closed states, respectively, in the presence of EF-G before translocation, in contrast with ΔSL-programmed PRE complexes, which sampled the hybrid/closed state approximately once before undergoing translocation. Quantitative analysis showed that the stimulatory stem loop destabilizes the hybrid state and elevates the energy barriers corresponding to subsequent substeps of translocation. The shift of the FSmRNA-programmed PRE complex equilibrium toward the classical/open state and toward states that favor EF-G dissociation apparently allows the PRE complex to explore alternative translocation pathways such as -1PRF.

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, w...

Here we use mechanical force to induce the unfolding and refolding of single RNA molecules: a simple RNA hairpin, a molecule containing a three-helix junction, and the P5abc domain of the Tetrahymena thermophila ribozyme. All three molecules (P5abc only in the absence of Mg 2+ ) can be mechanically unfolded at equilibrium, and when kept at constant force within a critical force range, are bi-stable and hop between folded and unfolded states. We determine the force-dependent equilibrium constants for folding/unfolding these single RNA molecules and the positions of their transition states along the reaction coordinate.

The 3'-end region of the genomic RNA of brome mosaic virus forms a tRNA-like structure that is critical for its replication. Previous studies have shown that in this region, a stem-loop structure, called SLC, is necessary and sufficient for the binding of the RNA replicase, and for RNA replication. Recently, we determined the high-resolution NMR structure of SLC, which demonstrated that a 5'-AUA-3' triloop region is an important structural element for the enzymatic recognition. We proposed that the 5'-adenine of the triloop, which is rigidly fixed ("clamped") to the stem, is a key recognition element for the replicase. To elucidate the role of this "clamped base motif" for the enzymatic recognition, we have now investigated the solution conformations of several stem-loop molecules with mutant triloops, 5'-UUA-3', 5'-GUA-3', 5'-CUA-3' and 5'-UUU-3', that destroy the enzymatic recognition. For the GUA and UUA mutants, we have obtained high-resolution solution structures using 2D NMR. All four mutants have very similar thermodynamic stabilities, and all have the same secondary structures, a triloop with a five base-paired stem helix. In addition, they have quite similar sugar puckering patterns in the triloop region. The NMR structures of the GUA and UUA show that the 5' nucleotide of the triloop (G6 in GUA or U6 in UUA) lacks the strong interactions that hold its base in a fixed position. In particular, the U6 of UUA is found in two different conformations. Neither of these two mutants has the clamped base motif that was observed in the wild-type. While UUA also shows global change in the overall triloop conformation, GUA shows a very similar triloop conformation to the wild-type except for the lack of this motif. The absence of the clamped base motif is the only common structural difference between these two mutants and the wild-type. These results clearly indicate that the loss of function of the UUA and GUA mutants comes mainly from the destruction of a small key recognition motif rather than from global changes in their triloop conformations. Based on this study, we conclude that the key structural motif in the triloop recognized by the replicase is a solution-exposed, 5'-adenine base in the triloop that is clamped to the stem helix, which is called a clamped adenine motif.

We determined the solution structure of two 27-nt RNA hairpins and their complexes with cobalt(III)-hexammine (Co(NH3)3+(6)) by NMR spectroscopy. The RNA hairpins used in this study are the P4 region from Escherichia coli RNase P RNA and a C-to-U mutant that confers altered divalent metal-ion specificity (Ca2+ replaces Mg2+) for catalytic activity of this ribozyme. Co(NH3)3+(6) is a useful spectroscopic probe for Mg(H2O)2+(6)-binding sites because both complexes have octahedral symmetry and have similar radii. The thermodynamics of binding to both RNA hairpins was studied using chemical shift changes upon titration with Mg2+, Ca2+, and Co(NH3)3+(6). We found that the equilibrium binding constants for each of the metal ions was essentially unchanged when the P4 model RNA hairpin was mutated, although the NMR structures show that the RNA hairpins adopt different conformations. In the C-to-U mutant a C.G base pair is replaced by U.G, and the conserved bulged uridine in the P4 wild-type stem shifts in the 3' direction by 1 nt. Intermolecular NOE cross-peaks between Co(NH3)3+(6) and RNA protons were used to locate the site of Co(NH3)3+(6) binding to both RNA hairpins. The metal ion binds in the major groove near a bulge loop, but is shifted 5' by more than 1 bp in the mutant. The change of the metal-ion binding site provides a possible explanation for changes in catalytic activity of the mutant RNase P in the presence of Ca2+.

The 3' end of brome mosaic virus RNA contains a tRNA-like sequence that directs its RNA synthesis. A stem loop structure in this sequence, stem loop C (SLC), was investigated using NMR, and correlated with its ability to direct RNA synthesis by its replicase. SLC consists of two discrete domains, a flexible stem with an internal loop and a rigid stem containing a 5'-AUA-3' triloop. Efficient RNA synthesis requires the sequence on only one side of the flexible stem and a specific compact conformation of the triloop. A high resolution structure of the triloop places the 5' adenine out in solution, and the 3' adenine within the triloop, held tightly through stacking and unusual hydrogen bonds. This high resolution structure of an RNA promoter from a (+)-strand RNA virus provides new insights into how the RNA-dependent RNA polymerase binds to the RNA to initiate synthesis.

We describe the RNA folding problem and contrast it with the much more difficult protein folding problem. RNA has four similar monomer units, whereas proteins have 20 very different residues. The folding of RNA is hierarchical in that secondary structure is much more stable than tertiary folding. In RNA the two levels of folding (secondary and tertiary) can be experimentally separated by the presence or absence of Mg2+. Secondary structure can be predicted successfully from experimental thermodynamic data on secondary structure elements: helices, loops, and bulges. Tertiary interactions can then be added without much distortion of the secondary structure. These observations suggest a folding algorithm to predict the structure of an RNA from its sequence. However, to solve the RNA folding problem one needs thermodynamic data on tertiary structure interactions, and identification and characterization of metal-ion binding sites. These data, together with force versus extension measurements on single RNA molecules, should provide the information necessary to test and refine the proposed algorithm.

The solution structure of an RNA hairpin modelling the P5 helix of a group I intron, complexed with Co(NH 3 ) 6 3+ , has been determined by nuclear magnetic resonance. Co(NH 3 ) 6 3+ , which possesses a geometry very close to Mg(H 2 O) 6 2+ , was used to identify and characterize a Mg 2+ binding site in the RNA. Strong and positive intermolecular nuclear Overhauser effect (NOE) cross-peaks define a specific complex in which the Co(NH 3 ) 6 3+ molecule is in the major groove of tandem G·U base-pairs. The structure of the RNA is characterized by a very low twist angle between the two G·U base-pairs, providing a flat and narrowed major groove. The Co(NH 3 ) 6 3+ , although highly localized, is free to rotate to hydrogen bond in several ways to the O4 atoms of the uracil bases and to N7 and O6 of the guanine bases. Negative and small NOE cross-peaks to other protons in the sequence reveal a non-specific or delocalized interaction, characterized by a high mobility of the cobalt ion. Mn 2+ titrations of P5 show specific broadening of protons of the G·U base-pairs that form the metal ion binding site, in agreement with the NOE data from Co(NH 3 ) 6 3+ . Binding constants for the interaction of Co(NH 3 ) 6 3+ and of Mg 2+ to P5 were determined by monitoring imino proton chemical shifts during titration of the RNA with the metal ions. Dissociation constants are on the order of 0.1 mM for Co(NH 3 ) 6 3+ and 1 mM for Mg 2+ . Binding studies were done on mutants with sequences corresponding to the three orientations of tandem G·U base-pairs. The affinities of Co(NH 3 ) 6 3+ and Mg 2+ for the tandem G·U base-pairs depend strongly on their sequences; the differences can be understood in terms of the different structures of the corresponding metal ion-RNA complexes. Substitution of G·C or A·U for G·U pairs also affected the binding, as expected. These structural and thermodynamic results provide systematic new information about major groove metal ion binding in RNA.

Identification and characterization of a metal ion binding site in an RNA pseudoknot was accomplished using cobalt (III) hexammine, Co(NH3)63+, as a probe for magnesium (II) hexahydrate, Mg(H2O)62+, in nuclear magnetic resonance (NMR) structural studies. The pseudoknot causes efficient -1 ribosomal frameshifting in mouse mammary tumor virus. Divalent metal ions, such as Mg2+, are critical for RNA structure and function; Mg2+preferentially stabilizes the pseudoknot relative to its constituent hairpins. The use of Co(NH3)63+as a substitute for Mg2+was investigated by ultraviolet absorbance melting curves, NMR titrations of the imino protons, and analysis of NMR spectra in the presence of Mg2+or Co (NH3)63+. The structure of the pseudoknot-Co(NH3)63+complex reveals an ion-binding pocket formed by a short, two-nucleotide loop and the major groove of a stem. Co(NH3)63+stabilizes the sharp loop-to-stem turn and reduces the electrostatic repulsion of the phosphates in three proximal strands. Hydrogen bonds are identified between the Co(NH3)63+protons and non-bridging phosphate oxygen atoms, 2' hydroxyl groups, and nitrogen and oxygen acceptors on the bases. The binding site is significantly different from that previously characterized in the major groove surface of tandem G.U base-pairs, but is similar to those observed in crystal structures of a fragment of the 5 S rRNA and the P5c helix of the Tetrahymena thermophila group I intron. Changes in chemical shifts occurred at the same pseudoknot protons on addition of Mg2+as on addition of Co(NH3)63+, indicating that both ions bind at the same site. Ion binding dissociation constants of approximately 0.6 mM and 5 mM (in 200 mM Na+and a temperature of 15 degrees C) were obtained for Co(NH3)63+and Mg2+, respectively, from the change in chemical shift as a function of metal ion concentration. An extensive array of non-sequence-specific hydrogen bond acceptors coupled with conserved structural elements within the binding pocket suggest a general mode of divalent metal ion stabilization of this type of frameshifter pseudoknot. These results provide new thermodynamic and structural insights into the role divalent metal ions play in stabilizing RNA tertiary structural motifs such as pseudoknots.

Coherences were observed between 15N3 of cytosine and its trans amino proton (H42) using a modified gradient-based heteronuclear single quantum coherence (HSQC) pulse sequence optimized for three-bond proton-nitrogen couplings. The method is demonstrated with a 22-nucleotide RNA fragment of the P5abc region of a group I intron uniformly labeled with 15N. Use of intraresidue 15 N3-amino proton couplings to assign cytosine 15 N3 signals complements the recently proposed JNN HNN COSY [Dingley, A.J. and Grzesiek, S. (1998) J. Am. Chem. Soc., 120, 8293–8297] method of identifying hydrogen-bonded base pairs in RNA.

The secondary structure of the P5abc subdomain (a 56-nt RNA) of the Tetrahymena thermophila group I intron ribozyme has been determined by NMR. Its base pairing in aqueous solution in the absence of magnesium ions is significantly different from the RNA in a crystal but is consistent with thermodynamic predictions. On addition of magnesium ions, the RNA folds into a tertiary structure with greatly changed base pairing consistent with the crystal structure: three Watson–Crick base pairs, three G⋅U base pairs, and an extra-stable tetraloop are lost. The common assumption that RNA folds by first forming secondary structure and then forming tertiary interactions from the unpaired bases is not always correct.

The structure of the L3 central hairpin loop isolated from the antigenomic sequence of the hepatitis delta virus ribozyme with the P2 and P3 stems from the ribozyme stacked on top of the loop has been determined by NMR spectroscopy. The 26 nt stem-loop structure contains nine base pairs and a 7 nt loop (5'-UCCUCGC-3'). This hairpin loop is critical for efficient catalysis in the intact ribozyme. The structure was determined using homonuclear and heteronuclear NMR techniques on non-labeled and15N-labeled RNA oligonucleotides. The overall root mean square deviation for the structure was 1.15 A (+/- 0.28 A) for the loop and the closing C.G base pair and 0.90 A (+/- 0.18 A) for the loop and the closing C.G base pair but without the lone purine in the loop, which is not well defined in the structure. The structure indicates a U.C base pair between the nucleotides on the 5'- and 3'-ends of the loop. This base pair is formed with a single hydrogen bond involving the cytosine exocyclic amino proton and the carbonyl O4 of the uracil. The most unexpected finding in the loop is a syn cytidine. While not unprecedented, syn pyrimidines are highly unusual. This one can be confidently established by intranucleotide distances between the ribose and the base determined by NMR spectroscopy. A similar study of the structure of this loop showed a somewhat different three-dimensional structure. A discussion of differences in the two structures, as well as possible sites of interaction with the cleavage site, will be presented.

Genetic information flows from DNA to RNA to protein. DNA is transcribed to yield messenger RNAs (the coding RNA), RNAs required for protein synthesis (ribosomal RNAs and transfer RNAs), and non-coding RNAs that often have regulatory functions (ncRNAs, including microRNAs, siRNAs, piRNAs,1 and others). Translation of messenger RNAs (mRNAs) produces the proteins that characterize the structures and functions of all organisms from bacteria to humans. At the core of the translation machinery is the ribosome, a macromolecular complex comprised of over 50 proteins and three ribosomal RNAs with a molecular weight of 2.5 MD for bacteria and 4.3 MD for humans.2 The ribosome cooperates with auxiliary species to ensure accurate and highly regulated translation. These species include initiation factors, elongation factors, transfer RNAs with attached amino acids, as well as release and recycling factors.3,4 In this review we will describe the application of single-molecule approaches to study the translation of mRNA by single ribosomes, focusing on mechanical manipulation with optical tweezers. Single-molecule methods have distinct advantages over traditional ensemble (or bulk) experiments in identifying the molecular mechanisms underlying this complex biological process. This has particularly important implications for elucidating the kinetics of processive molecular machines, such as ribosomes. The kinetics contain abundant information about how molecular machines act in a coordinated fashion to accomplish a complicated task, such as deciphering a nucleic acid sequence and synthesizing the encoded protein. However, kinetics are governed by stochastic processes; thus, each molecule in a reaction takes a different amount of time to react. It is impossible to maintain synchronicity over several steps of a sequential reaction. At any time in an ensemble reaction of many molecules, there will be reactants, products, and all the intermediates. For a large number of ribosomes that start translating a particular mRNA at the same time, some will be decoding the first codon while others are reading the second, third, fourth, etc. In contrast, at any time in a single-molecule reaction there is only one species. The characteristics of each single species can be determined. Optical tweezers have been used to study translation by a single ribosome on one mRNA. In the first published study, constant force was applied to the 3’- and 5’-ends of a harping RNA.5 As the ribosome translated the hairpin, double strand RNA was converted to single strands, thus increasing the end-to-end distance of the RNA molecule. The increase in distance directly measures the translocation of the ribosome. In a subsequent set of experiments, force is applied to either the 3’- or 5-end of the RNA while holding on to the ribosome. This geometry allows studying the ribosome as a motor under assisting force or opposing force as it moves along its track (the mRNA). A third geometry has been developed in which force is applied across the nascent polypeptide. This experimental setup will allow synthesis of the translation product to be monitored. 1.1 The Machinery of Translation High resolution structures of the ribosomal subunits6-12 as well as the 70S ribosome13 have been obtained by X-ray diffraction around the year 2000. A thorough review (with over 80 references) of the structural aspects of the functions of the ribosome is given at the Nobel Prize 2009 website (http://www.nobelprize.org/nobel_prizes/chemistry/laureates/2009/advanced.html). Further details are described by each Nobelist in their published Nobel lectures.14-16 The structures of bacterial ribosomes complexed with mRNAs, tRNAs, initiation, elongation, and release factors, and antibiotics are presented. More recent atomic resolution structures include a 3.0 A structure of a yeast eukaryotic ribosome,17-19 the human and Drosophila 80S ribosome 20, release factor 2 and 3 bound to a bacterial ribosome,21-23 and EF-G bound to the ribosome.24-27 Cryo-electron microscopy, cryo-EM, provides further information about different states of the ribosome and its interactions with external factors.28-31 Structural information of many of the auxiliary species, free and bound to the ribosome are now available: initiation factors, eIF1;32 elongation factors, EF-G,33,34 EF-Tu,35-39, as well as release and recycling factors.40-43

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.

Metal ions that link phosphates and guanine bases are necessary for the folding of an RNA enzyme. A magnesium ion core may be a common feature of RNA structure.

Background: Solvated metal ions are critical for the proper folding and function of RNA. Despite the importance of these ions, the details of specific metal ion–RNA interactions are poorly understood. The crystal structure of a group I intron ribozyme domain characterized several metal-binding sites in the RNA with osmium (III) hexammine bound in the major groove. A corresponding method for locating and characterizing metal-binding sites of RNA in solution is of obvious interest. NMR should be ideal for localizing metal hexammine ions bound to the RNA because of the large concentration of protons around the metal center. Results: We have solved the solution structure of the P5b stem loop from a group I intron ribozyme bound to a cobalt (III) hexammine ion. The location of the ion is precisely determined by intermolecular nuclear Overhausser effect cross-peaks between the cobalt (III) hexammine protons and both exchangeable and non-exchangeable RNA protons in the major groove. The binding site consists of tandem G–U base pairs in a sequence of four consecutive G residues ending in a GAAA tetraloop, as originally identified in the crystal structure. The edges of the bases in the major groove present an electrostatically negative face and a variety of hydrogen-bond acceptors for the cobalt (III) hexammine ion. The metal ion ligand is bound near the guanosine nucleotides of the adjacent G–U base pairs, where it makes hydrogen bonds with the N7 and carbonyl groups of both guanines. The carbonyl groups of the uracil residues add to the negative surface of the binding pocket, but do not form hydrogen bonds with the hexammine. Additional hydrogen bonds form with other guanine residues of the GGGG sequence. The structure of the binding site does not change significantly on binding the cobalt (III) hexammine. The structure of the complex in solution is very similar to the structure in the crystal. Conclusions: The structure presents a picture of how tandem G–U base pairs bind and position metal ions within the RNA major groove. The binding site is performed in the absence of metal ions, and presents a negative pocket in the major groove with a variety of hydrogen-bond acceptors. Because G–U base pairs are such a common motif in RNA sequences, it is possible that this RNA–metal ion interaction is critical in forming large complex RNA structures such as those found in the ribosome and self-splicing introns. This structure was determined using cobalt (III) hexammine as an analog for hexahydrated magnesium, a technique that may be applicable to other RNA sequences. Metal hexammines may prove to be useful general probes for locating RNA metal ion binding sites in solution.

RNA molecules fold into specific base-paired conformations that contain single-stranded regions, A-form double helices, hairpin loops, internal loops, bulges, junctions, pseudoknots, kissing hairpins, and so forth. These structural motifs are recognized by proteins, other RNAs, and other parts of the same RNA. The interactions of these structural elements are crucial to the biological functions of the RNA molecules. We describe the different motifs and discuss their thermodynamic stabilities relative to single strands of RNA. The stabilities determine under what conditions they occur and whether they change when interacting with proteins or other ligands.