Review Focus On Function: Single Molecule RNA Enzymology

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ReviewReviewFocus on Function: Single Molecule RNA EnzymologyFocus on Function: Single Molecule RNA EnzymologyMark A. Ditzler,1 Elvin A. Alemán,2 David Rueda,2 Nils G. Walter31Biophysics Research Division, Single Molecule Analysis Group, University of Michigan, Ann Arbor, MI 481092Department of Chemistry, Wayne State University, Detroit, MI 482023Department of Chemistry, Single Molecule Analysis Group, University of Michigan, Ann Arbor, MI 48109Received 3 July 2007; revised 24 July 2007; accepted 24 July 2007Published online 8 August 2007 in Wiley InterScience ( DOI 10.1002/bip.20819ABSTRACT:The ability of RNA to catalyze chemical reactions wasfirst demonstrated 25 years ago with the discovery thatThis article was originally published online as an acceptedpreprint. The ‘‘Published Online’’ date corresponds to thepreprint version. You can request a copy of the preprint byemailing the Biopolymers editorial office at biopolymers@wiley.comgroup I introns and RNase P function as RNA enzymesA(ribozymes). Several additional ribozymes wereINTRODUCTIONsubsequently identified, most notably the ribosome,detailed understanding of the biopolymer ribonucleic acid (RNA) is of great importance throughoutthe life sciences. RNA-coding genes are now recognized to be far more abundant in eukaryotes thantheir protein-coding counterparts and are essentialto the central biochemical processes within all living cells.1–3RNA is responsible for the synthesis of all proteins within thecell, plays a central role in replication of many viruses, regulates gene expression in both bacteria and eukaryotes, isinvolved in the maintenance, processing, modification, andediting of genetic information, and probably carries out a hostof still unknown cellular processes. The discovery of the catalytic capabilities of group I introns4 and RNase P,5 coupledwith the knowledge that certain viral genomes are composedentirely of RNA, established RNA as unique in nature for itsability to both store genetic information and catalyze chemicalreactions. The dual genetic and catalytic role of RNA lends tremendous support to the hypothesis that purely RNA-basedlife predated the emergence of both protein and DNA.6–8 Inaddition to their important functions in nature, catalyticRNAs have been used to derive RNA-based therapeutics.9,10Our understanding of the molecular underpinnings of organisms, and possibly the origin of life, as well as the developmentof new medicines, therefore, significantly depend on our ability to dissect the fundamental properties of RNA enzymes.Naturally occurring ribozymes can be divided into severalgroups based on their size: small self-cleaving RNAs (\200nucleotides), medium-sized self-splicing introns, and largerfollowed by intense mechanistic studies. More recently,the introduction of single molecule tools has dissected thekinetic steps of several ribozymes in unprecedented detailand has revealed surprising heterogeneity not evidentfrom ensemble approaches. Still, many fundamentalquestions of how RNA enzymes work at the molecularlevel remain unanswered. This review surveys the currentstatus of our understanding of RNA catalysis at the singlemolecule level and discusses the existing challenges andopportunities in developing suitable assays. # 2007 WileyPeriodicals, Inc. Biopolymers 87: 302–316, 2007.Keywords: single molecule microscopy; fluorescenceresonance energy transfer; ribozyme; ribosome; catalyticRNACorrespondence to: Nils G. Walter; e-mail: or David Rueda;e-mail: rueda@chem.wayne.eduContract grant sponsor: National Institutes of HealthContract grant number: GM62357Contract grant sponsor: Wayne State UniversityC 2007V302Wiley Periodicals, Inc.Biopolymers Volume 87 / Number 5–6

Single Molecule RNA EnzymologyFIGURE 1 Reaction mechanism of the two ribozymes highlightedhere. (A) Site-specific phosphodiester transfer as catalyzed by theself-cleaving small ribozymes, including the hairpin ribozyme. Asuitably positioned base B deprotonates the 20 -OH of the upstreamribose, thereby activating the 20 -oxygen for nucleophilic in line attackon the scissile phosphodiester. The 50 -oxygen leaving group is protonated by a properly positioned acid AH1. (B) Peptide bond formation as catalyzed by the ribosome. A suitably positioned base deprotonates the amino acid esterified with the A-site tRNA, therebyactivating the amino group for nucleophilic attack on the peptidyltRNA ester bond on the P-site tRNA. The 30 -oxygen leaving group isprotonated by a properly positioned acid AH1.catalytic ribonuclear-protein (RNP) complexes. The class ofsmall ribozymes comprises the hairpin, hammerhead, hepatitis delta virus (HDV), Varkud satellite (VS), and glmSribozymes. All of these ribozymes catalyze a site-specific RNAbackbone cleavage reaction as well as the reverse ligation reaction. Cleavage is achieved through an SN2-like reaction mechanism in which the 20 -hydroxyl (20 -OH) of the cleaved strandacts as the nucleophile, resulting in 20 ,30 -cyclic-phosphateand 50 -OH termini on the 50 - and 30 -products, respectively(Figure 1A).11–13 On the other end of the spectrum, largeRNPs such as RNase P, the spliceosome, and the ribosome represent catalytic RNAs that recruit protein cofactors for optimal function in vivo (self-splicing introns are of intermediatecomplexity as some of them require protein cofactors andothers do not). RNase P and the spliceosome carry out sitespecific hydrolysis and transesterification reactions on RNAbackbones, respectively, through mechanisms distinct fromBiopolymers DOI 10.1002/bip303that of the small ribozymes. The ribosome is unique amongthe naturally occurring ribozymes in that it generates a product that is not itself an RNA. The ribosome catalyzes peptidebond formation between amino acids coupled to tRNA adapters and so is responsible for the production of all cellular protein (Figure 1B). Evidence that the RNA rather than proteincomponents of RNPs are catalytic stems from the observationof catalytic competence in the absence of protein and/or anactive site composed of RNA only.4,5,14,15Since their discovery a quarter-century ago, extensiveinvestigations into the catalytic mechanisms of ribozymeshave been conducted in the quest to understand and potentially exploit this essential and ubiquitous class of enzymes.Until recently, catalytic RNAs were studied in bulk solution,where the number of molecules present is many orders ofmagnitude larger than the low copy number typical of manyRNAs and RNPs in a single cell (1–103, up to 106 in case ofthe ribosome). Recently, it has become increasingly commonto study protein and RNA enzymes using single moleculemethods, offering the ability to observe short-lived mechanistic intermediates and minor subpopulations often maskedin the ensemble average. Single molecule approaches tounderstanding RNA include atomic force microscopy, opticaltweezers, and single molecule fluorescence microscopy (forreview please see Refs. 16 and 17). Of these, single moleculefluorescence resonance energy transfer (smFRET) has provenparticularly effective in studying reaction pathways of ribozymes, as smFRET assays provide information on the globaldynamics of molecules under native conditions. smFRET hastherefore provided researchers with the unique opportunityto quantify the (equilibrium) kinetics of both directions inreversible reactions, which are commonly found in RNA.In this review we first survey the insights gained fromsingle molecule probing of catalysis by two representativeribozymes and focus on structural dynamics as a signaturefor catalysis. We then discuss the bottleneck presented by theneed to develop suitable assays that probe specific steps on areaction pathway, as well as proven or plausible routes toovercoming this obstacle to the broader use of single molecule techniques. Single molecule studies of RNA foldingpathways have been thoroughly reviewed elsewhere.17–19EXAMPLES OF SINGLE MOLECULEENZYMOLOGYCurrently, the primary approach used in single moleculeRNA enzymology is to monitor global conformationalchanges associated with individual steps on or off a reactionpathway such as substrate binding, tertiary structure(un)folding, chemical catalysis, and product release. In thefollowing we will explore in detail single molecule investiga-

304Ditzler et al.tions that highlight the scope and limitations of single molecule RNA enzymology. We will focus on two significant RNAcatalysts at opposite ends of the spectrum, the hairpin ribozyme and the ribosome. The hairpin ribozyme is probably themost investigated RNA in single molecule enzymology. Theribosome is far more complex and has been subjected to fewersingle molecule studies than the comparably simple hairpinribozyme. However, the tremendous biological importance ofprotein biosynthesis has motivated substantial progress alsoon single molecule enzymology of the ribosome.The Hairpin Ribozyme: Synergy Between SingleMolecule and Ensemble AssaysThe hairpin ribozyme (Figure 2A) is a small noncoding RNAthat facilitates site-specific cleavage and ligation chemistry ofits own backbone as part of the double-rolling circle replication of Nepovirus satellite RNAs. It serves as a convenientmodel system to study RNA catalysis, and a vast body of ensemble biochemical,20–26 structural,27–33 and computationaldata34,35 is available, as are extensive single molecule analyses.36–42 The drive toward a complete understanding of catalysis in this system has demonstrated and exploited the powerof single molecule spectroscopy to uncover short-lived intermediates, minor subpopulations, and molecular heterogeneity, which otherwise are all hidden in the ensemble average.An effective application of single molecule techniques, conversely, requires correlation of statistically significant averagesfrom stochastic single molecule events with observables fromensemble measurements. In fact, most successful approacheshave relied on the availability of a thorough characterizationFIGURE 2 Single molecule FRET applied to hairpin ribozyme docking. (A) A two-stranded(RzA, RzB) hairpin ribozyme binds substrate (orange and small letters; arrow, cleavage site) toform internal loops A and B, each flanked by two helices (H1–H4) and connected between H2 andH3. Noncanonical base pairs are indicated by dashed lines. Tertiary structure docking occurs via ag11:C25 Watson–Crick base pair (red), a ribose zipper (blue), and the U42 binding pocket(purple). Terminal Cy3 and Cy5 fluorophores serve as donor/acceptor FRET pair and biotin is usedfor surface immobilization through binding to streptavidin. (B) Multistep reaction pathway of thehairpin ribozyme with distinct kinetic steps identified by their rate constants. (C) Typical smFRETtime trajectory monitoring donor and acceptor emission intensity, together with the resultingFRET 5 IA/(ID 1 IA) trace. Characteristic of a single molecule observation are the anticorrelateddonor and acceptor signals and the single-step photobleaching; specific events are indicated. Rateconstants are calculated from statistically significant numbers of state dwell times and corrected asdescribed.36,42 (D) Two FRET time trajectories from different molecules show dramatically differentdwell times in the high-FRET docked state that reveal persistent heterogeneity between molecularsubpopulations. Reproduced from Ref. 36, with permission from American Association for theAdvancement of Science.Biopolymers DOI 10.1002/bip

Single Molecule RNA Enzymologyof ensemble behavior in order to interpret single moleculeobservations with confidence.In the case of the hairpin ribozyme, extensive insightsfrom ensemble techniques into the ribozyme’s structural andkinetic properties have formed a solid platform for probingat the single molecule level. For example, ensemble FRETexperiments in solution revealed the existence of two structural states at equilibrium—the catalytically inactiveundocked and the active docked conformations.20 Upondocking, the internal loops of domains A and B are broughtinto close contact, compacting the RNA (Figure 2A).43,44Several crystallographic studies showed that this docked stateis stabilized by a number of well-characterized tertiary hydrogen bond and base-stacking interactions (Figure 2A).30–33 Inaddition, ensemble enzymology approaches were appliedextensively, yet the (presumably microreversible) mechanismof cleavage and ligation remains debated.45 Nucleobasederived general acid–base catalysis,21,30 water assisted acid–base catalysis,33,34 and transition state charge stabilization22–24,31,46have all been invoked as possible mechanisms. Theimportant contributions that a single nucleobase or even afunctional group can make to proper RNA folding as well ascatalysis42 and the inherent ambiguity in the interpretationof enzymologic results45 contribute to the difficulty of pinpointing the reaction mechanism and necessitate additionalmechanistic probing tools.smFRET based on biotin-streptavidin-mediated surfaceimmobilization and total internal reflection fluorescence microscopy has been employed to dissect the reaction pathway ofthe hairpin ribozyme, which comprises substrate binding,interdomain docking, substrate cleavage, interdomainundocking, and finally product release (Figure 2B). By labeling the termini of the two interacting domains with a suitablesmFRET donor/acceptor pair such as cyanine dyes Cy3/Cy5,47the docked, undocked, and product/substrate-free (unbound)states of the ribozyme display distinguishable FRET levels(defined as IA/(ID 1 IA), where IA and ID are the fluorescencesignals from acceptor and donor, respectively) (Figures 2 and3A).36 Single-step photobleaching to background signal at theend of each smFRET time trajectory confirms that indeed asingle RNA molecule is observed (Figure 2C). Since the cleavage products rapidly dissociate from the undocked state,cleavage and subsequent undocking result in a decrease insmFRET from the docked to the unbound state (Figure 3A).The good agreement between the rate of unbound stateappearance in smFRET and that of product appearance asmonitored by traditional (ensemble) electrophoretic separation further supports the assignment of states and the functional validity of single molecule trajectories (Figure 3A).36Biopolymers DOI 10.1002/bip305FIGURE 3 Accessing reaction chemistry of the hairpin ribozymethrough single molecule FRET. (A) smFRET time trace of the twoway junction form of the hairpin ribozyme, showing the docked,undocked, and substrate/product free states at distinct FRET values.36 The purple box and bar indicate equivalent processes on thereaction scheme and the experimental data, respectively. Individualstates are indicated as U (undocked), D (docked), L (ligated), C(cleaved), and P (product). (B) smFRET time trace of the four-wayjunction form of the hairpin ribozyme, illustrating the difference in(un)docking dynamics before and after cleavage (i.e., in the ligatedand cleaved forms). The purple box and bar highlight equivalentprocesses on the reaction scheme and in the experimental data,respectively.39 (C) Schematic of the possible outcome scenariosfrom a double buffer-exchange experiment (first removal, thenreplenishment of 12 mM Mg21) on the two-way junction form ofthe hairpin ribozyme with the associated experimental smFRETreadouts.49 Reproduced from Refs. 36,39, and 49, with permissionfrom American Association for the Advancement of Science, NaturePublishing Group, and National Academy of Sciences.To determine (un)docking rate constants in the intactribozyme–substrate complex unaffected by cleavage, a blocking 20 -O-methyl substitution was introduced into the active

306Ditzler et adenosine (A-1), based on its preservation of the sugarpucker preference and hydrogen bond acceptor capacity ofthe native 20 -OH.36 Substrate dissociation is slow understandard conditions (pH 7.5, 12 mM Mg21), effectively isolating the docking/undocking steps from the remaining reaction pathway. Similarly, (un)docking rate constants in theisolated ribozyme-product complex can be determined byinstalling a ligation blocking 30 -phosphate on the 50 -productinstead of the natural 20 ,30 -cyclic phosphate. Rate constantscan then be extracted by plotting the cumulative number,N(t), of state dwell (residence) times that are shorter thantime t and fitting with a multiexponential of the form:N ðtÞ ¼XAi ð1 e ki;obs t Þð1ÞiThe observed rate constants ki,obs need to be corrected forphotobleaching, which shortens the observed dwell times;while the amplitudes Ai need to be corrected if indeed multiple rate constants are observed, to avoid bias toward shorterdwell times.36,42 Corrected dwell times in the docked statethen determine the undocking rate constant(s) kundock,whereas corrected dwell times in the undocked state separately determine the docking rate constant(s) kdock (Figure2B). This ability to determine forward and reverse rate constants of a reversible reaction independently of each other isan important advantage of single molecule enzymology. Inthe ensemble average, only collective and synchronized relaxation of many molecules from one state to another can beobserved, yielding an aggregate rate constant.The fact that single molecule data are often best represented through multiple rate constants ki,obs is another distinction from ensemble averaging techniques. In the hairpinribozyme (and commonly in RNA), an underlying molecularheterogeneity is apparent upon closer inspection (Figure2D).36,37,40 At least four distinct subpopulations of moleculesare found in individual smFRET time trajectories,36,37,42 eachof which undocks with one of the four rate constantsextracted from the docking dwell times. Representatives ofeach subpopulation are remarkably resistant to interconversion as they continue to exhibit the same undocking behavioreven when probed at 3-h intervals at 258C,36 in variousMg21 concentrations,37 or in the presence of various RNAmodifications.42 Such static heterogeneity (or molecularmemory) is also observed in the context of a four-way junction form of the ribozyme, with potentially even more subpopulations exhibiting heterogeneity in both docking andundocking kinetics,38 and is independent of the strategy forRNA surface immobilization.40 The four distinctly undocking molecular subpopulations map onto fast and slow phasesof biphasic ensemble cleavage42 and folding assays,25 and sohave important consequences for the interpretation of datafrom ensemble measurements.The Hairpin Ribozyme: Accessing ChemistryAlthough docking and undocking rate constants of the hairpinribozyme can be derived directly from dwell time analyses ofsmFRET trajectories, catalysis itself does not result in any discernable change in smFRET signal and thus calls for less directinference. Three different approaches have been pursued so far.In the first approach, single molecule probing of inactivatedribozyme–substrate and –product complexes is combinedwith ensemble cleavage assays and classic mechanistic modeling.36,42 Essentially, the intrinsic cleavage and ligation rate constants are derived by finding either a numerical36 or analyticalsolution42 to the set of differential equations that defines thereaction pathway in Figure 2B after substrate binding (wheresubstrate binding is assumed to be irreversible):8SdNundock SS ¼ kdock Nundockþ kundock Ndock dt S dNdock pSS dt ¼ kdock Nundock ðkundock þ kcleav ÞNdock þ klig Ndock PdNdockppSPP¼ kcleav Ndock ðklig þ kundockÞNdockþ kdock Nundock dt P dNundock PPPP ¼ kundockNdock ðkdockþ koff ÞNundockþ kon Ndiss dt dNdiss P:¼ koff Nundock kon Ndissdtð2Þwhere N denotes the population of molecules in a given statewith the subscript indicating the conformation (docked orundocked) and the superscript indicating whether the ribozyme is bound to substrate (S) or product (P). Using theknown overall cleavage kinetics and assuming the completeabsence of interconversion between the molecular subpopulations throughout the catalytic cycle (which makes them independent of one another), a numerical fit yields upper andlower bounds for the intrinsic cleavage and ligation rate constants and an estimate of their ratio.36 Alternatively, Eqs. 2can be formulated in matrix notation so that an analyticalsimulation of the overall cleavage time course is derived bydiagonalizing and solving the corresponding master equation.42 If the chemical equilibrium constant is independentlydetermined by, for example, running a ligation reaction tocompletion in the presence of excess reaction product, theproblem of solving the master equation for the five unimolecular reactions that describe the kinetic pathway is reduced to asingle-variable fit.42 The use of substrate and product analogsBiopolymers DOI 10.1002/bip

Single Molecule RNA Enzymologyand matrix-algebra assisted kinetic simulations thus enablesrapid relative comparison of single functional group variants.It was discovered that functional groups far from the dockinginteractions and active site directly impact both docking andchemistry. These findings led to the proposal that, similar toprotein enzymes,48 long-range coupled molecular motionsexist in ribozymes that link the overall fold to the active siteand contribute to RNA function.42 Recent molecular dynamics (MD) simulations support this hypothesis.34It is important to note that two additional assumptions areimplicitly employed in this analysis. First, common intrinsiccleavage and ligation rate constants are assumed for all molecular subpopulations so that the derived rate constants represent averages over all molecules (a standard feature also of ensemble enzymology). A range of rate constants may exist, butkinetic modeling suggests that the chemical rate constantsvary by less than threefold between the different subpopulations.42 Second, in the analysis it is assumed that the dockingand undocking rate constants obtained for the inactivatedribozyme complexes (with 20 -O-methyl modified substrateand 30 -phosphate modified 50 -product analogs) closelyresemble those of the active complexes. Recent evidence fromsingle molecule studies in the presence of cleavable substratesuggests that undocking of the substrate complex is decelerated and that of the product complex accelerated in the presence of the native 20 -OH and 20 ,30 -cyclic phosphate, respectively, leading to a systematic overestimation of the intrinsiccleavage rate constant in the earlier analysis by approximatelysevenfold (while docking and ligation rate constants are unaffected; see also the following discussion).39,49The second single molecule approach used to accesschemical rate constants of the hairpin ribozyme exploits thefact that undocking is slow in the native substrate complex,but fast in the cleaved product complex; thus, cleavage in situcan be fortuitously detected by an acceleration of thesmFRET fluctuations between docked and undocked conformations (rather than a change in FRET level).39 This changein dynamics is particularly pronounced at 1 mM Mg21 in afour-way junction form of the ribozyme, in which both the50 - and 30 -products are extended to prevent dissociation. Significant enhancement and suppression of the docking/undocking kinetics can thus be used as signatures for cleavage and ligation, respectively (Figure 3B). Still, extraction ofa cleavage rate constant is complicated since, first, any giventransition from docked to undocked state may originatefrom either the substrate or product complex; second, cleavage events followed by ligation before undocking will goundetected; and, third, the observed docked state dwell timesare shortened by photobleaching. Using a succession ofundocking events as indication of cleavage and correcting forBiopolymers DOI 10.1002/bip307missed events as well as photobleaching then yields an estimate of the intrinsic cleavage rate constant. In addition, theligation rate constant has to be corrected for the fact that arapidly docking/undocking ribozyme spends only part of itstime in the docked state where ligation can occur. The finalcorrected intrinsic chemistry rate constants indicate a stronger equilibrium bias toward the ligated state than did the useof chemistry blocking modifications.39The third approach exploited to tease out the intrinsicchemistry rate constants that also uses cleavable substrate andsets up a succession of buffer exchanges to produce distincttime sequences of smFRET signal that serve as kinetic ‘‘fingerprints’’ of specific catalytic intermediates.49 In concept, suchan approach is analogous to pulse-chase experiments widelyused in ensemble enzymology, but it gains from the ability toassign a specific state to each individual molecule and countthe number of representatives. Figure 3C illustrates how thenumber of molecules in the undocked (U) and docked (D)states in the presence of either ligated (L) or cleaved (C) substrate is assessed. First, chemical equilibrium is reached uponincubation of the ribozyme in standard buffer (pH 7.5, 12mM Mg21) in the presence of a saturating excess of 30 -product. Upon addition of EDTA to remove Mg21 at time t0 andsubsequent replenishment of Mg21 at time t1, distinct scenarios are observed depending on which of the four reactionintermediates UL, DL, DC, or UC are observed. In particular,the two docked states undock (transit from high [0.8] to low[0.2] FRET) upon Mg21 removal, while the two undockedstates only slightly decrease in FRET (from 0.3 to 0.2). Inaddition, the two complexes involving ligated substrate willeventually dock again after the replenishment of Mg21,whereas the two cleaved complexes lose their 30 -productunder these conditions (the 50 -product is covalently linked tothe ribozyme) and thus can never dock after Mg21 addition.Given a sufficient observation window after time t1 (i.e., slowphotobleaching), the four reaction intermediates can beunequivocally identified through their unique FRET versustime patterns (Figure 3C) and counted. Based on a sufficientnumber of molecule assignments P, yielding P(UL) 5 21,P(DL) 5 591, P(DC) 5 47, and P(UC) 5 165, the equilibriumconstants of docking before and after cleavage and of internalchemistry are derived as ratios of the appropriate moleculecounts.49 In conjunction with the rate constant of the verylast transition from high to low FRET under standard conditions (Figure 3A), which is a convolution of the undocking,cleavage, and ligation rate constants, the intrinsic cleavageand ligation rate constants can be calculated.The latter two single molecule studies yield similar intrinsic cleavage and ligation rate constants and a consistent picture of how a ribozyme is optimized for its self-cleavage

308Ditzler et al.function in Nepovirus replication—stable docking of theligated ribozyme–substrate complex allows for ample time tocleave, while instable docking of the cleaved ribozyme–product complex results in rapid product release.39,49 One maynote that a structural explanation for this kinetic phenomenon is still outstanding. In addition, since a properly ligatedRNA is important as a replication template, one may wonderwhether alternating structures switch the RNA between preferably cleaved (active) and ligated (inactive) forms. Strikingly, standard ensemble measurements of the chemistryequilibrium position do not distinguish between the dockedand the undocked ligated or cleaved states and thus lead to asignificant underestimation of the ligation equilibrium constant (from [P(UL) 1 P(DL)]/[P(DC) 1 P(UC)] 2.9)42compared to its true value (which is defined as P(DL)/P(DC) 13),49 and a resulting overestimation of the intrinsic cleavage rate constant. Ensemble studies also average out important information on parallel (heterogeneous) reaction pathways, which are studied in isolation when observing singlemolecules, and on short-lived intermediates, which are identified by short smFRET bursts as long as they live longer thanthe experimental time resolution (Figure 2D).In summary, single molecule enzymology studies of thehairpin ribozyme have demonstrated feasible routes towarddetermining rate and equilibrium constants of the chemicalstep in a fully reversible RNA reaction pathway that exhibitsmolecular heterogeneity. It thus has become possible to dissect the often surprisingly profound role(s) of individual residues and functional groups in structural dynamics andchemistry, may they be close to or far from the active siteand/or tertiary structure interactions. Careful considerationneeds to be given to the various types of modifications (fluorophore labeling, surface immobilization, functional group,and sequence changes) that have to be introduced into theRNA to address specific scientific questions. Powerful synergies arise from the use of multiple alternate approaches.Future advances in our understanding of the mechanism ofsite-specific backbone cleavage will require a careful integration of single molecule fluorescence approaches with thoseof, in particular, ensemble enzymology coupled with mutagenesis, X-ray crystallography, NMR spectroscopy, MDsimulation, and quantum mechanical/molecular mechanicalcalculations.The Ribosome: A Complex RNA-Protein MachineryThe largest ribozyme studied so far at the single moleculelevel is the protein biosynthetic machinery, the ribosome,arguably the most abundant enzyme on earth. Ribosomesare very large (in bacteria 2.5 MDa) RNA-protein com-plexes that universally translate the sequence of a messengerRNA (mRNA) with high fidelity into a polypeptide chainusing transfer RNA (tRNA) adaptors. Ribosomes are composed of a large and a small subunit (termed 50S and 30S inbacteria, respectively). Translation is initiated by the assembly of the two subunits into the 70S ribosome on an mRNAtemplate. Protein synthesis is catalyzed by the ribosomalRNA (rRNA) component of the large subunit by transfer ofthe growing peptide chain from one tRNA onto the nextaminoacyl-tR

Focus on Function: Single Molecule RNA Enzymology Mark A. Ditzler,1 Elvin A. Alema n,2 David Rueda,2 Nils G. Walter3 1 Biophysics Research Division, Single Molecule Analysis Group, University of Michigan, . editing of genetic information, and probably carries out a host of still unknown cellular processes. The discovery of the cata-

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