Mechanism of RNA Translocation by a ViralPackaging Motor

JI Í LÍSAL

Institute of Biotechnology and Department of Biological and Environmental Sciences

Division of BiochemistryFaculty of Biosciences

University of Helsinki andViikki Graduate School in Biosciences

ACADEMIC DISSERTATION

To be presented, with permission of the Faculty of Biosciences of the University ofHelsinki, for public criticism in the auditorium 1041 of Biocenter 2, Viikikaari 5,

Helsinki, on September 15th, 2006, at 12 noon.

Helsinki 2006

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Supervisor

Docent Roman TumaInstitute of Biotechnology andDepartment of Biological and Environmental SciencesUniversity of HelsinkiFinland

Reviewers

Professor Nynke H. DekkerKavli Institute of NanoscienceDelft University of TechnologyThe Netherlands

Professor Lloyd W. RuddockBiocenter Oulu and Department of BiochemistryUniversity of OuluFinland

Opponent

Professor Peter E. Prevelige, Jr.Department of MicrobiologyUniversity of Alabama at BirminghamUSA

Cover art - The P4 hexameric motor in the act of packaging ssRNA into emptycapsid. A 20 Å icosahedral reconstruction of 6 procapsid is shown in a cyanisosurface representation (de Haas et al. 1999). The density for one of the P4 turretshas been removed and substituted with a ribbon representation of the crystal structureof hexameric P4 from bacteriophage 12 (Mancini et al. 2004a). A modeled A-formss-RNA is shown. Courtesy of Dr. Erika Mancini, Oxford University.

© Jiri Lisal 2006

ISBN 952-10-3351-7 (paperback)ISBN 952-10-3352-5 (PDF, http://ethesis.helsinki.fi)

ISSN 1795-7079

Yliopistopaino, Helsinki University Printing HouseHelsinki 2006

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Original publications

This thesis is based on the following articles, which are referred to in the text by theirRoman numerals.

I. Jiri Lisal, Denis E. Kainov, Dennis H. Bamford, George J. Thomas, Jr., andRoman Tuma (2004): Enzymatic Mechanism of RNA Translocation inDouble-stranded RNA Bacteriophages. J. Biol. Chem. 279: 1343 - 1350.

II. Jiri Lisal and Roman Tuma (2005): Cooperative mechanism of RNApackaging motor. J. Biol. Chem. 280: 23157-64.

III. Jiri Lisal, TuKiet T. Lam, Denis E. Kainov, Mark R. Emmett, Alan G. Marshalland Roman Tuma (2005): Functional visualization of viral molecular motorby hydrogen-deuterium exchange reveals transient states. Nat. Struct. Mol.Biol. 12: 460-466.

IV. Jiri Lisal, Denis E. Kainov, TuKiet T. Lam, Mark R. Emmett, Hui Wei, PaulGottlieb, Alan G. Marshall and Roman Tuma (2006): Interaction of packagingmotor with the polymerase complex of dsRNA bacteriophage. Virology. 351:73-79.

V. Jiri Lisal and Roman Tuma (2006): RNA binding and translocation by acooperative molecular motor at single molecule level. Manuscript.

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Abbreviations

ADP adenosine diphosphateAMP adenosine monophosphateAMP-CPP adenosine 5’-( -methylene)triphosphateAMP-PNP adenosine 5’-( -imido)triphosphateATP adenosine triphosphateATPase adenosine triphosphataseb basebp base pairDNA deoxyribonucleic acidds double-strandedEM electron microscopyFRET fluorescence resonance energy transferFT-ICR Fourier transform-ion cyclotron resonancegp gene productHDX hydrogen-deuterium exchangeHPLC high performance liquid chromatographyMANT methylanthraniloylNA nucleic acidNDP nucleoside diphosphateNMP nucleoside monophosphateNMR nuclear magnetic resonanceNTP nucleoside triphosphateNTPase nucleoside triphosphatasePC procapsidPi inorganic phosphatePPi inorganic pyrophosphatePoly(C) polycytidylic acidPX polymerase complexRMSD root mean square deviationRNA ribonucleic acidSV40 LTA simian virus 40 large tumor antigenss single-strandedTNP trinitrophenylTPM tethered particle motionwt wild type

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Summary

Molecular motors are proteins that convert chemical energy into mechanicalwork. The viral packaging ATPase P4 is a hexameric molecular motor thattranslocates RNA into preformed viral capsids. P4 belongs to the ubiquitous class ofhexameric helicases. Although its structure is known, the mechanism of RNAtranslocation remains elusive. Here we present a detailed kinetic study of nucleotidebinding, hydrolysis, and product release by P4. We propose a stochastic-sequentialcooperative model to describe the coordination of ATP hydrolysis within thehexamer. In this model the apparent cooperativity is a result of hydrolysis stimulationby ATP and RNA binding to neighboring subunits rather than cooperative nucleotidebinding. Simultaneous interaction of neighboring subunits with RNA makes theotherwise random hydrolysis sequential and processive.

Further, we use hydrogen/deuterium exchange detected by high resolutionmass spectrometry to visualize P4 conformational dynamics during the catalytic cycle.Concerted changes of exchange kinetics reveal a cooperative unit that dynamicallylinks ATP binding sites and the central RNA binding channel. The cooperative unit iscompatible with the structure-based model in which translocation is effected byconformational changes of a limited protein region. Deuterium labeling also disclosesthe transition state associated with RNA loading which proceeds via opening of thehexameric ring.

Hydrogen/deuterium exchange is further used to delineate the interactions ofthe P4 hexamer with the viral procapsid. P4 associates with the procapsid via its C-terminal face. The interactions stabilize subunit interfaces within the hexamer. Theconformation of the virus-bound hexamer is more stable than the hexamer in solution,which is prone to spontaneous ring openings. We propose that the stabilization withinthe viral capsid increases the packaging processivity and confers selectivity duringRNA loading.

Finally, we use single molecule techniques to characterize P4 translocationalong RNA. While the P4 hexamer encloses RNA topologically within the centralchannel, it diffuses randomly along the RNA. In the presence of ATP, unidirectionalnet movement is discernible in addition to the stochastic motion. The diffusion ishindered by activation energy barriers that depend on the nucleotide binding state.The results suggest that P4 employs an electrostatic clutch instead of cycling throughstable, discrete, RNA binding states during translocation. Conformational changescoupled to ATP hydrolysis modify the electrostatic potential inside the centralchannel, which in turn biases RNA motion in one direction. Implications of the P4model for other hexameric molecular motors are discussed.

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Table of contents

Original publications

Abbreviations

Summary

Table of contents

A. INTRODUCTIONA.1. Molecular motors

A.1.1. Cytoskeletal motorsA.1.2. Rotary motors

A.2. Motors associated with nucleic acidsA.2.1. PolymerasesA.2.2. HelicasesA.2.3. Viral packaging motors

A.3. Packaging motors of CystoviridaeA.3.1. Role of the packaging motor in Cystoviridae life cycleA.3.2. Structure of the packaging motor P4

A.4. Dynamics of a molecular motor studied by hydrogen-deuteriumexchange

A.5. Single molecule techniques as tools to study molecular motors

B. AIMS OF THE STUDY

C. MATERIAL AND METHODS C.1. Reagents and preparations C.2. Data analysis and software tools

D. RESULTS AND DISCUSSIOND.1. ATP binding, hydrolysis, and ADP releaseD.2. RNA bindingD.3. RNA translocationD.4. Mechanism of single translocation stepD.5. Mechanism of step coordinationD.6. P4 activity regulation within the polymerase complexD.7. Implications for other systems

E. CONCLUSIONS AND FUTURE PROSPECTS

F. ACKNOWLEDGEMENTS

G. REFERENCES

Reprints of original publications

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A. INTRODUCTION

A.1. Molecular motors

Physical movement is one of themain attributes of life. On a macroscopicscale, animals and microbes move to findfood and to avoid poisons and predators.Plants turn their leaves toward theirenergy source, sunlight. Especially on amicroscopic scale, movement isabsolutely essential for life. There is acontinuous transport of organelles andmolecules across the cell. Molecules areselectively transported between differentcell compartments. Incredibly complexand orchestrated movements of cellcomponents are required for celldivision. All these forms of movementrely on microscopic machines calledmolecular motors. A molecular motor isa protein which converts chemical (orelectrochemical) energy into mechanicalwork (Howard 2001). All molecularmotors move in steps. One step isusually associated with one completecatalytic cycle of the molecular motor.Most of the molecular motors catalyzehydrolysis of ATP into ADP andinorganic phosphate (Pi) in order topower their mechanical cycle. In vivo,under physiological conditions, the freeenergy that is stored in the terminal ( -)-anhydride bond of an ATP molecule is

about -9×10-20 J, equaling about -50 kJper mol of ATP, depending on thespecific cellular conditions. Study of thecomplete mechanochemical cycle isessential in order to fully understand howa molecular motor functions.

There are two theoreticalconcepts that have been proposed for themolecular motor mechanisms. (i) The“Power stroke” mechanism assumes thateach catalytic cycle leads to a sequenceof conformational changes that shifts themotor exactly one step forward. Themotor works in a deterministic fashionsimilarly as do macroscopic motors. (ii)

The presence of unavoidable thermalfluctuations on the microscopic scale ledto a concept of a “Brownian motor”(Astumian 1997). The Brownian motoruses thermal fluctuation from itssurroundings to facilitate its movement.Due to stochastic nature of thermalfluctuations, a Brownian motor movesstochastically in both directions.However, the forward steps are morefrequent because the chemical energyreleased by the catalytic cycle puts biason the system. The limiting case of theBrownian motor is the so-called“Brownian ratchet”. Locally, theBrownian ratchet moves with equalprobability in both directions; howeversome of the complete forward steps arerectified by the chemical energy releasedby the catalytic cycle.

In this work I will delineate theprinciples of the mechanism of amolecular motor P4 which packagesgenomic RNA into preformed viralparticles. In order to do that it is useful tosummarize the principles that have beenderived from studies of other molecularmotors.

A.1.1. Cytoskeletal motorsInternal order in a eukaryotic cell

is maintained in part by molecularmotors that shuttle organelles andmolecules along cytoskeletal tracksconsisting of self-assembling proteinslike tubulin and actin (Schliwa 2003).There are three types of cytoskeletalmotors – kinesins, myosins and dyneins.These were the first protein complexes tobe acknowledged to work as themolecular motors. All of them are multi-protein complexes that are able toachieve movement by binding andhydrolyzing adenosine-5’-triphosphate(ATP) within a globular motor domain.

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One or two motor domains are usuallycoupled to an extended tail whichmediates binding of different cargo.

Kinesin translocates towards theplus end of microtubules. It contains twomotor domains that move in a hand overhand manner, taking 8 nm long steps(Svoboda et al. 1993). The step sizematches the periodicity of themicrotubules. Conventional kinesin is

able to produce forces up to 6 pN(Meyhofer and Howard 1995). Bindingof ATP to the motor domain isassociated with a conformational changeleading to translocation along themicrotubule. ATP hydrolysis is coupledto release of kinesin from themicrotubule allowing for a recoverystroke while ADP release leads again tothe tight microtubule binding (Fig. 1A).

Figure 1. Schematic model of hydrolysis cycle for single-headed kinesin (A) and myosin(B). The nucleotide binding state (E – empty binding site, T – ATP binding, DP – ADP and Pibinding, D – ADP binding) controls conformation of the motor and its binding to thecytoskeletal track. According to (Howard 2001).

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The motor domain of kinesin isstructurally related to that of myosin.Myosin moves unidirectionaly alongactin filaments. Myosin II, which isresponsible for skeletal musclecontraction, exhibits a catalytic cycle thatis qualitatively similar to that of kinesin(Fig. 1B). Myosin II moves in 5.3 nmsteps (length of actin monomer)(Kitamura et al. 1999) and is able togenerate forces up to 10 pN (Ishijima etal. 1996). The double headed myosin Vmoves in 36 nm steps (length of actinrepeat unit) and is able to generate forcesup to 4 pN. The 36 nm step can befurther resolved into 12 and 24 nm longsubsteps (Uemura et al. 2004). The 12nm substep occurs after ATP binding tothe actin-bound trailing head and the 24nm substep results from an isomerizationof the actin-bound leading head in theADP state.

Dynein is structurally differentfrom the two motors discussed above.Dynein structure resembles that ofAAA+ enzymes (Burgess et al. 2003;Vallee and Hook 2003). Dynein movestowards the minus end of microtubuleand also plays an important role in themotion of eukaryotic flagella. Dyneinseems to change the step size with load(Mallik et al. 2004). At loadsapproaching the stalling force of 1.1 pNdynein moves in a 8 nm steps (matchingthe periodicity of the microtubules). Atlower loads dynein may move also inmultiples of this basic step i.e. in 16 nmand 24 nm steps.

In summary, all three cytoskeletalmotors move in steps of constant lengthwhich is given by the periodicity of thetrack. Due to limited amount of energyobtained from the ATP hydrolysis cycle,motors with the long step generate lowerforces than motors with the short step.Efficiency of the energy conversion bythe cytoskeletal motors is very high incomparison with the efficiency of themacroscopic motors, reaching 60 to80%. The periodicity of RNA, the track

for P4 translocation, is 0.3 nm. Thus, inanalogy with the cytoskeletal motors, P4is expected to move in 0.3 (or its smallmultiple) nm-long steps. In theory, sucha short step should allow P4 to exertforces on the order of 100 pN.

A.1.2. Rotary motorsCurrently, the best understood

molecular motor is F0F1-ATP synthase.Results obtained for this rotary motor arerelevant to P4 mechanism because theF1-ATPase exhibits sequence andstructural similarity to P4 (Kainov et al.2003b; Mancini et al. 2004a). Thus, wewill review the mechanism of F1-ATPasein detail.

The F0F1-ATP synthase is foundin all organisms where it uses energyprovided by the flow of protons down anelectrochemical gradient and across thecorresponding membrane (the plasmamembrane in bacteria, the mitochondrialmembrane in eukaryotes and thethylakoid membrane of chloroplasts inplants) to keep the reaction ATP ADP+ Pi far from equilibrium, favoring ATPsynthesis. F0F1-ATP synthase consists oftwo interdependent molecular motors, F0and F1. Membrane-embedded F0 isassembled from three differentpolypeptides a, b, and c with thestoichiometry of one a, two b and 9-12copies of subunit c per functional unit(Fig. 2). The associated F1 consists offive subunit types ( through withthree copies each of the large subunitsand and one copy each of the minorsubunits , and (Fig. 2). Proton flowthrough F0 subunits a and c causesrotation of these subunit relative to eachother. The c-ring of F0 is coupled to the-subunit of F1. Thus, the -subunit

rotates relative to the / stator andpowers ATP synthesis at the /interfaces of F1. Under specificconditions (e.g. in the lysozomalmembrane or in vitro) the F1 may alsohydrolyze ATP resulting in protonpumping across the membrane. Hence

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this molecular machine can run inreverse.

Six nucleotide binding sites arelocated on the / interfaces of F1. Thethree binding sites associated with thesubunits are catalytically active. Theyshow an extraordinary degree of positivecatalytic cooperativity. When all of thethree sites are filled with substrate,catalysis is more than 105 times fasterthan if only one substrate is bound(Senior et al. 2002). A ‘binding changemechanism’ as the basis of the ATPsynthesis has been proposed (Boyer1993). According to this hypothesis,each of the catalytically active nucleotidebinding sites is in a different structuraland functional state at any given timeduring catalysis. During sequentialcatalytic cycles, all of the sites passthrough each of these conformationaland functional states. While one of thesites is void of substrates (ADP and Pi),the second site has both substrates boundand the third site is in a conformationthat promotes bond formation betweenADP and Pi, resulting in elimination ofwater and the synthesis of ATP (Fig. 3).The binding change mechanism furtherstates that the energy for the synthesis ofATP is solely required for the release ofthe product from the active site and not

for the actual formation of the ( - )-anhydride bond.

An X-ray crystallographic modelof the beef heart mitochondrial ATPase(Abrahams et al. 1994) gives detailedinformation about the structure of thebinding sites. Two of the catalytic sitesare rather similar in conformation, whilethe third site differs significantly fromthe others in structure. This third sitedoes not have nucleotide bound, and apart of the binding site is rotated into anopen conformation that facilitates theproduct release. The sequential change inthe conformation of catalytic sites isdriven by rotation of the asymmetric -subunit inside the central cavity of the

/ ring. The structure of F1 in complexwith ADP·AlF4¯ (Menz et al. 2001) addsa detailed view of the catalytic transitionstate with all three catalytic sitesoccupied by nucleotides.

Rotation of the –subunit hasbeen observed directly for single F1motors in the ATP hydrolyzing mode(Noji et al. 1997). These studies revealedthree 120° steps in the rotation, eachcorresponding to the hydrolysis of oneATP molecule. Calculated from therotational velocity, the torque reachedduring rotation is >40 pN nm. At lowATP concentrations even smallersubsteps of 90° and 30° were resolved(Yasuda et al. 2001; Nishizaka et al.2004). They are associated with ATPbinding and ADP release, respectively.The –subunit rotation during protonpowered ATP synthesis in the completeF0F1-ATP synthase was also measured(Diez et al. 2004). A three-step rotationwas observed. The direction of rotationduring ATP synthesis was opposite tothe direction observed during ATPhydrolysis.

On the molecular level, therotation of -subunit during ATPhydrolysis is driven by largeconformational changes of the -subunits. The 90° rotation is associatedwith ATP binding. Initial binding of

Figure 2. Schematic model of the bacterialF0F1-ATP synthase. The front subunit ofthe F1 ring is removed.

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ATP is accomplished by the formation ofmultiple weak bonds. As bindingprogresses, the catalytic site closes upand pulls the top part of the -subunittoward the bottom part. The constanttorque can be explained by sequentialincrease in the number of bonds (the‘binding zipper’ hypothesis (Antes et al.2003; Oster and Wang 2003)). After theATP-driven conformational change, theATP is stably bound and the reactiontransiently stops. Subsequent insertion ofan “arginine finger” from a neighboring

-subunit into the ATP-binding site is

necessary for ATP hydrolysis(Nadanaciva et al. 1999; Dittrich et al.2004). This is likely to be the cause ofthe observed positive cooperativity. ATPhydrolysis per se does not contributemuch to work output. However, theproduct release following the hydrolysisallows the 30° rotation to proceed. Thismakes the 120° step complete.

The F0 motor is less wellunderstood but its mechanism seems toshare similar principles with that ofbacterial flagellar motor (Berry 2000;Blair 2003; Oster and Wang 2003).

Figure 3. Scheme of the binding change mechanism. Rotation of subunit (oval with anarrow in the center of the cartoon) drives sequential conformational changes of nucleotidebinding sites and thus changes their affinity for different nucleotides. According to (Boyer1993).

A.2. Motors associated with nucleic acids

An important class of themolecular motors encompasses motorsassociated with nucleic acids. This classof molecular motors includes enzymessuch as polymerases and helicases, andalso the object of our study, thepackaging motor P4. Motors of this classtranslocate along nucleic acids whileperforming number of tasks essential forreplication, maintenance, and expressionof genetic information. In addition, theribosome could belong to this class ofmolecular motors because it translocatesalong mRNA molecules (Vanzi et al.2003). The polymerases, helicases and

viral packaging motors will be describedin more details in the following sections.

A.2.1. PolymerasesTranscription and replication of

genetic material are among the mostfundamental processes that constitute theessence of life. Replication of DNA isaccomplished by DNA polymerases thatsynthesize copies of genomic DNA withhigh fidelity, while the first step in theexpression of genetic informationencoded in DNA is performed by RNApolymerases. Both kinds of enzymescatalyze the same type of reaction –

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template-directed polymerization ofnucleoside triphosphates (NTPs).

From a mechanistic point ofview, polymerases can be seen asmolecular motors that performdirectional mechanical work againstdissipative forces during polymerization.Such forces are hydrodynamic drag andimpediments imposed by specificsequences encoded in DNA as well as bynumerous proteins that hold onto DNAsuch as histones or transcription factors(Schliwa 2003).

Polymerases are so far the bestunderstood NA-associated motors thathave been studied widely not only bystandard ensemble techniques but alsoon single molecule level using opticaltweezers (Yin et al. 1995; Wang et al.1998; Davenport et al. 2000; Wuite et al.2000; Shaevitz et al. 2003; Thomen etal. 2005). Experiments at the singlemolecule level constitute practically theonly way how to measure properties of,and forces generated by, individualunsynchronized molecular motors. Adetailed description of some of thesingle molecule techniques is given laterin this thesis. Although P4 is related tohelicases rather than to polymerasesthere is a paucity of single moleculeresults for the former class of motors.Thus, the results obtained forpolymerases provide a useful frameworkfor understanding other NA associatedmotors and P4 in particular.

The single molecule studies ofpolymerases have shown that nearlyevery polymerization step is coupled tothe forward translocation step of theenzyme. However, transcriptional pausesand backsliding occur occasionally.Transcriptional pauses are usuallyassociated with specific DNA sequenceswhile backslidings result from theincorporations of a wrong nucleotidethat is consequently removed. RNApolymerases exhibit a polymerizationrates 40-50 nucleotides/s and are able to

move against loads up to 14-25 pN (Yinet al. 1995; Wang et al. 1998). SomeDNA polymerases reach polymerizationrates of 500-1000 nucleotides/s andexhibit stalling forces as high as 34 pN(Wuite et al. 2000).

Recently, an observation ofsingle base steps in transcriptionalelongation has been made(Abbondanzieri et al. 2005).Observation of stepping velocities atdifferent ATP concentrations andopposing forces limited the number ofpossible models for RNApolymerization mechanism. Theexperimental data were inconsistent witha model for movement incorporating apower stroke tightly coupled topyrophosphate release. Instead, theresults favored a Brownian ratchetmodel incorporating a secondary NTPbinding site (Fig. 4), a mechanism that isfurther supported by structural data(Westover et al. 2004).

A.2.2. HelicasesThe object of our study, the viral

packaging motor P4, exhibitsunambiguous sequence and structuralsimilarity to hexameric helicases(Kainov et al. 2003b; Mancini et al.2004a). A helicase is a molecular motorthat uses the energy of NTP hydrolysisto unidirectionally translocate along aNA strand and to separate (unwind) thecomplementary strand of the NA duplex(von Hippel and Delagoutte 2001). Atphysiological conditions, the dsNA base-pairs open and close spontaneously(Delagoutte and von Hippel 2002;Schliwa 2003). As the helicase movesalong and binds to the ssNA, it preventsrebinding of the complementary strand.In a similar fashion helicases can alsodestabilize secondary structure of RNA,remove NA associated proteins, andthread NA through various pores.

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Figure 4. Alternative kinetic models for RNA polymerase translocation. (A) A powerstroke model in which translocation ( , red) is driven by pyrophosphate (PPi) release. (B) ABrownian ratchet model where reversible oscillation between pre- and post-translocationstates occurs before NTP binding (blue). (C) A Brownian ratchet model where translocationand NTP binding can occur in either order. This model postulates the existence of a secondaryNTP site to accommodate the possibility of nucleotide binding when the enzyme is in its pre-translocated state. With permission from (Abbondanzieri et al. 2005).

Helicases are necessary for DNAreplication, repair, recombination,transcription, ribosome biogenesis,translation, RNA splicing, RNA editing,RNA transport, RNA degradation,bacterial conjugation and viralpackaging/unpackaging (Lohman andBjornson 1996; von Hippel andDelagoutte 2001; Caruthers and McKay2002; Delagoutte and von Hippel 2002;Schliwa 2003). Approximately 2% ofeukaryotic genes code for helicases.Mutations in genes coding for helicasesresult in several human diseasesincluding cancer and premature aging.

The oligomeric state constitutes abasis for helicase classification into twolarge groups: (i) ring (hexameric)helicases and (ii) non-ring (monomericand dimeric) helicases (Patel and Picha2000). Each helicase subunit has a singleNTP binding site and a distinct NAbinding site. NTPase activity modulates

the NA affinity, and vice versa. Inmonomeric helicases the modulation ofthe NA affinity together withconformational changes of the helicasemay lead to translocation along NAstrand using a Brownian motormechanism as proposed recently for theHCV helicase NS3 (Levin et al. 2005)(Fig. 5).

Many hexameric helicases (e.g.T7 gp4 and Rho transcriptionterminator) exhibit a two-stage NAbinding (Ahnert et al. 2000; Kim andPatel 2001; Skordalakes and Berger2003) (Fig. 6). The NA binds to aprimary binding site which is located onthe perimeter of the ring, and inducesring opening. Subsequently, the NA slipsinto a secondary binding site which islocated inside the central channel. Thisis followed by ring closure and NAunbinding from the primary binding site.

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Hexameric helicases (includingP4) possess six identical NTP bindingsites. In analogy with F1-ATPase,cooperativity between the binding sitesis expected. A model suggesting a‘binding change’ mechanism in whichNTP hydrolysis proceeds sequentiallyaround the hexamer has been proposedon the basis of the T7 gp4 helicasestructure (Singleton et al. 2000). In thismodel, NTP binding, hydrolysis, andproduct release were proposed to triggermovements of subunits relative to eachother. The subunit movements wouldthen effect DNA translocation throughthe central channel of the T7 gp4hexamer.

A completely different model forhexameric helicases has been based on aset of structures of the SV40 LTAhelicase (Gai et al. 2004). The modelproposes that NTP binding, hydrolysisand product release are concerted andproceed at the same time at all sixsubunits. Associated conformationalchanges constitute re-orientations of thedomains within a subunit, creating an‘iris’-like motion in the hexamer.Additionally, six hairpins that protrudeinto the central channel movelongitudinally along the channel,possibly pulling DNA through thechannel.

So far there is no consensuswhich of the models is/are correct orwhether they apply also to otherhexameric helicases. My goal in thiswork is to elucidate the mechanism ofP4 which belongs to the class ofhexameric helicases. Comparison of theP4 mechanism with those proposedabove shall reveal commoncharacteristics of the hexameric helicasemotor function.

Figure 5. Brownian motor mechanism ofHCV helicase translocation along ssDNA.The binding free energy ( G) of thehelicase ssDNA complex (gray circles)changes along the length of the DNA (solidlines) and the sawtooth profile is shaped byspecific interactions in the complex. In theabsence of ATP, the helicase binds ssDNAtightly and settles into a deep energyminimum unlikely to move (position 1).ATP binding changes the properties of theDNA binding site, and the interactionsbecomes weaker and almost constant alongDNA (position 2). In this transient state, thehelicase moves along the DNA randomlyand bidirectionally. Rapid hydrolysis of thebound ATP restores the asymmetric sawtooth profile of the complex and forces thehelicase to slide down the energy slope.Depending on the direction of the netrandom movement during the previousstage, the helicase lands either in the sameminimum (position 1) or one step ahead(position 3). Binding of the new ATPmolecule allows the helicase to start the nexttranslocation cycle (position 4). Accordingto (Levin et al. 2005).

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Figure 6. Scheme of the Rho transcription terminator ring opening mechanism.RNA binds to a primary binding site (b). The primary binding induces ring opening(c). The RNA strand slips into a secondary binding site which is located inside thecentral channel (d). Finally, the ring closes and RNA is released from the primarybinding site (e).

A.2.3. Viral packaging motorsA virus is a (lipo)protein particle

carrying inside its own geneticinformation (in the form of DNA orRNA). Viruses are not able to replicatethemselves. For successful replication, avirus needs to deliver its geneticinformation into a host cell. The viralgenetic information is expressed withinthe host cell to produce new viralproteins and to replicate the viralgenome. In some viruses, the newlyproduced viral NA condenses while it iscoated by the newly produced viralproteins. In other viruses (e.g. in tailedbacteriophages or herpes simplex virus),an empty protein shell (procapsid) isformed first. The procapsid is then filledby the viral NA in an energy-consumingprocess called “NA packaging”, which isperformed by a viral “packaging motor”(Fig. 7) (Moore and Prevelige 2002;Catalano 2005; Ponchon et al. 2005).

The only packaging motor forwhich detail mechanochemicalcharacterization has been achieved isthat of Bacillus subtilis bacteriophage

29. This dsDNA packaging motorconsists of three components: (i) thehead-tail connector, a dodecamer of

protein p10, which is attached to aunique five-fold prohead vertex, (ii) amultimeric ring of prohead RNA(pRNA), which is attached to theconnector and (iii) several copies ofATPase gp16 that binds to pRNA(Catalano 2005). A highly speculativemechanism of the motor mechanismincluding rotation of the connector hasbeen based on the connector structureand the symmetry mismatch between theconnector and the prohead (Simpson etal. 2000). Mechanochemical studies(using an optical trap) showed that the

29 packaging motor is able to generateforces up to 75 pN (Smith et al. 2001).DNA translocation is likely triggered byphosphate release and the motor subunitsact in a coordinated, successive fashionwith high processivity (Chemla et al.2005).

The P4 hexamer is considerablysimpler than, and structurally differentfrom, the 29 packaging motor, asdescribed in the following section.Therefore, the mechanism of P4 is likelyto differ from that of the 29 packagingmotor.

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Figure 7. A generic dsDNA bacteriophage capsid assembly pathway. Here an enzymecalled ‘terminase’ acts as the packaging motor. With permission from (Steven et al. 2005).

Figure 8. Simplified scheme of the Cystoviridae core replication. The ssRNA transcriptsare labeled l+, m+, and s+. The dsRNA genome segments are labeled L, M, and S. Withpermission from (Kainov et al. 2004).

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A.3. Packaging motors of Cystoviridae

A.3.1. Role of the packaging motor inCystoviridae life cycledsRNA bacteriophages 6- 14 from theCystoviridae family primarily infect theplant pathogenic bacteriumPseudomonas syringae (Semancik et al.1973; Mindich et al. 1999). TheCystoviridae virion consists of threestructural layers: the outermost protein-lipid envelope, a nucleocapsid proteinshell (which is missing in 8(Hoogstraten et al. 2000)) and the innericosahedral polymerase complex (PX).The outer layers facilitate interactionswith the host cell and are sequentiallyremoved during virus entry while the PXis delivered into the cytoplasm (Bamfordet al. 1976). The PX is a multifunctionalmachine which performs genomeencapsidation, replication andtranscription. PX is composed of 120copies of the major structural protein P1(Ktistakis and Lang 1987), about 12monomers of RNA-dependent RNApolymerase (Makeyev and Bamford2000), 12 hexamers of packaging motorP4 (Juuti et al. 1998) and about 30dimers of assembly factor P7 (Juuti andBamford 1997). P2 and P4 reside at thefive-fold vertices of the icosahedralPX(de Haas et al. 1999; Ikonen et al.2003). The PX contains ~13 kb dsRNAgenome composed of three segments: L,large (~6 kb); M, middle (~4 kb); and S,small (~3 kb) (Semancik et al. 1973).The non-coding regions at the 3’ and 5’–ends of the genome segments containsignals for replication, packaging andtranscription of the genome (Mindich1999; Pirttimaa and Bamford 2000).

The Cystoviridae life cycle isschematically depicted in Fig. 8. Uponinfection, the dsRNA genome moleculesare transcribed by P2 inside the PXproducing mRNAs that are extrudedfrom the PX. The packaging motor P4 isrequired during the transcription(Pirttimaa et al. 2002) to act as a passive

pore for mRNAs extrusion (Kainov et al.2004). The mRNAs are translated usinghost cell ribosomes. The newlysynthesized viral proteins P1, P2, P4 andP7 self-assemble into a procapsid (PC)(Poranen et al. 2001; Kainov et al.2003a). In the 6, P4 nucleates the PCassembly. The viral mRNAs arerecognized by binding sites on P1, and asingle copy of each segment is packagedsequentially into the PC by thepackaging motor P4. P4 acts as an activeNTP-ase during the packaging (Frilanderand Bamford 1995; Paatero et al. 1995).The sequential order of packaging isachieved by PC expansion duringpackaging of the S segment that exposesa high affinity binding site for the Msegment. Similarly, when the M segmentis packaged, the M sites disappear and Lsites appear (Qiao et al. 1997; Mindich1999). When the packaging of the Lsegment is completed, ssRNA genomeprecursors are replicated inside the PXto yield genomic dsRNAs (Frilander etal. 1992). The PX can then performeither additional rounds of transcriptionor mature into infectious virions.

A.3.2. Structure of the packagingmotor P4The packaging motor P4 is a hexameric,ssRNA-stimulated NTPase (Juuti et al.1998; Kainov et al. 2003b). P4 sequenceexhibits all the motifs (H1-H4) that arecharacteristic of the DnaB helicase(Patel and Picha 2000). P4s frombacteriophages 8 and 13 translocatealong ssRNA in 5’ to 3’ direction whilepossessing helicase activity in vitro(Kainov et al. 2003b). The structure ofthe P4 hexamer from bacteriophage 12(Mancini et al. 2004a) (Fig. 9) ishomologous to those of other DnaB-likehexameric helicases. The triangularwedge-shaped P4 monomer (35 kDa) iscomposed of an N-terminal domain, acentral ATPase RecA-like core which is

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conserved among DnaB-like hexamerichelicases, and a C-terminal region(Mancini et al. 2004a). The sequence ofthe N-terminal domain is not conservedbetween different members ofCystoviridae and may be involved infunctions other than RNA translocation.The central core, together with part ofthe C-terminal region, forms aRossmann-type nucleotide-bindingdomain containing a twisted, eight-stranded sheet flanked by fivehelices. Six P4 monomers associate intoa symmetric hexamer, enclosing acentral channel, through which RNA istranslocated. Outer diameter of the P4hexameric ring is ~95 Å and a height~55 Å. The width of the funnel-likecentral channel varies from 25 Å to 21Å. Six equivalent nucleotide-bindingsites are located at the interfacesbetween adjacent subunits. In the crystalstructure of P4 with the substrate analog(AMP-CPP-Mg2+) or with ADP, all sixnucleotide-binding sites were occupiedby the corresponding nucleotide(Mancini et al. 2004a).

Comparison of P4 structures indifferent nucleotide states revealed thattwo regions of P4 subunit undergosignificant conformational changes as aconsequence of ATP hydrolysis andphosphate release (Mancini et al.2004a). One of the regions is the socalled “P-loop” which interacts withand phosphates of the boundnucleotide and coordinates the Mg2+ ion.In the complex of P4 with a substrateanalog, the P-loop is in a “relaxed”conformation, whereas in the productcomplex, it folds into a “strained”conformation (Fig. 10). Theconformation of the P-loop correlateswith the position of RNA binding site(helix 6 and loop L2) which protrudesinto the central channel. The RNAbinding site moves from an “up”position in the substrate complex to a“down” position in the product complex

Figure 9. Structure of the P4 hexamer.The P4 hexamer is shown in top (N-terminal domain), side, and bottom (C-terminus) views. The secondary structuralelements are colored according to the barwhere different colors distinguishsubdomains or segments of the P4monomer: N-terminal safety pin motif(blue), all domain (dark purple),conserved RecA-like ATP binding domain(red), and antiparallel strands and C-terminal helix (green). Six molecules ofAMPcPP, drawn in ball-and-stickrepresentation, are located in the bindingsites between monomers. With permissionfrom (Mancini et al. 2004a).

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It was proposed that this movement isassociated with a ~6 Å translocation of

the bound RNA (Mancini et al. 2004a).

Figure 10. P4 conformational changes accompanying nucleotide hydrolysis. Thestructures of one P4 subunit in complex with AMPcPP-Mg2+ (A) and ADP-Mg2+ (B),respectively, are compared. 6 helix - purple, L2-loop cyan, P-loop - red, AMPcPP - orange,ADP - blue, Mg2+ ion - pink sphere. With permission from (Kainov et al. 2006).

A.4. Dynamics of a molecular motor studied by hydrogen-deuteriumexchange

X-ray diffraction provides staticsnapshots of protein structure within theconstraints of the crystal state. Whilevery valuable as such, it can give onlylimited information about proteindynamics and the structures of transientstates. A suitable method that canprovide such information under nativeconditions is amide hydrogen-deuteriumexchange (HDX). Amide hydrogensprovide individual probes along theentire protein sequence. The rate ofamide HDX of a given protein regionreflects its solvent accessibility andstructural stability. NMR-basedmeasurement of amide hydrogenexchange has been used widely tocharacterize the folding and stability ofrelatively small, soluble proteins

(Englander et al. 1996). The use of massspectrometry for HDX detectionextended the range of accessible systemsto high molecular mass proteins andassemblies of limited solubility oravailability (Lanman and Prevelige2004). Here we apply HDX for the firsttime to study the dynamics andquarternary structure changes of amolecular motor.

In a typical HDX experiment(Fig. 11) a protein of interest is dilutedinto an excess of heavy water (D2O).The exchange reaction is sampled at thedesired times (seconds to days) andquenched by lowering the pH to 2.5 andthe temperature to 0°C. The protein isthen nonspecifically digested into shortpeptides by pepsin or another acidic

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protease. The buffer salts are removedand peptides partially separated by rapidreverse phase HPLC. The deuteriumcontent in each peptide is then analyzedusing high resolution mass spectrometry.

The observed rate kobs of HDXdepends on the chemical exchange ratekch which is given by the local chemicalenvironment of the given amidehydrogen as well as by the globalconditions such as pH and temperature.HDX is also markedly affected byprotein flexibility and mobility.Fluctuations in protein structure allowtransient solvent exposure even ofdeeply buried areas. These structuralfluctuations can be described by rateconstants for opening and closing, kopand kcl. The observed HDX rate is thengiven by a general formula:kobs=(kop·kch)/(kcl+kch+kop) (Hoofnagle et

al. 2003). In the case of fast fluctuations,represented by native state fluctuation orprotein “breathing” motions, whenkcl>>kch and kcl>>kop the formulasimplifies to kobs=(kop·kch)/kcl which istermed the EX2 limit. In this limit onecan not observe directly the rate ofstructural fluctuations but only thechanges in equilibrium between the openand the closed state. In the case of slowstructural changes such as global proteinfolding/unfolding when kop and kcl<<kch,the observed HDX rate is equal to therate of structural opening kobs=kop. Thisis termed the EX1 limit. In this case onecan directly measure the rate of thestructural change kop on the time scale ofthe experiment (seconds to days in atypical HDX MS experimental setup).However, measurement of a specificconformational change within the motor

Figure 11. Scheme of a typical HDX experiment. Parts of the HPLC, including theinjection syringe, solvent precooling loop, sample loop, injector, and capillary column are allimmersed in ice to minimize back-exchange.With permission from (Hoofnagle et al. 2003).

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cycle requires synchronization of themotors e.g. by rapid addition of a ligandwhich promotes the conformationalchange. Here we aim to use HDX

method to study the dynamics andquarternary structure changes of P4during RNA binding and throughout theATP hydrolysis cycle.

A.5. Single molecule techniques as tools to study molecular motors

Ensemble measurements yieldonly average values. In contrast, single-molecule experiments allow one toexamine individual members of aheterogeneous population and toidentify, sort and compare theirsubpopulations (Weiss 1999). Thus,single molecule experiments may revealnew information about enzymes with acomplicated catalytic cycle, such asmolecular motors, that are difficult tosynchronize in ensemble. In its simplestversion, a single-molecule experimentrequires immobilization of the molecularmotor (or its track) and labeling of themotor (or its moving part) with a brightfluorophore that can be used to followthe motion by microscope imaging. Thissimplest approach was used to confirmand study the rotation of the F1 ATPase-subunit (Noji et al. 1997). Observation

of cytoskeletal motor translocation with1.5 nm resolution was recently achievedusing extremely photostablefluorophores and computer analysis ofthe collected images (FIONA -fluorescence imaging with one-nanometer accuracy) (Yildiz et al.2004).

Nanometer scale conformationalchanges of molecular motors can bedetected using fluorescence (Förster)resonance energy transfer (FRET) thatrelies on distance-dependentnonradiative energy transfer between adonor and acceptor fluorophores (Selvin1995). FRET at the single molecule levelwas used, for example, to studyconformational changes (Rasnik et al.2004) and translocation (Myong et al.2005) of the Rep helicase, or to study

rotation in F0F1-ATP synthase (Diez etal. 2004).

Translocation of a molecularmotor along NA can be studied by atethered particle motion method (TPM)(Yin et al. 1994; Pouget et al. 2004).The TPM method has been widely usedto study dsDNA motors such aspolymerases (Schafer et al. 1991; Yin etal. 1999; Tolic-Norrelykke et al. 2004),RecBCD helicase (Dohoney and Gelles2001) or RuvAB complex (Dennis et al.2004). Here we use the TPM method tostudy passive diffusion and activetranslocation of P4 along ssRNA at zeroforce. The TPM method employs directobservation of the restricted Brownianmotion of a microsphere that isconnected to a surface immobilizedmolecular motor via a single NAmolecule, the tether. The molecularmotor changes the tether length and thusthe amplitude of the microsphere motionwhich is directly visualized bymicroscope.

Forces generated by a singlemolecular motor may be measured bymany techniques includingmicropipettes, atomic force microscopy(AFM), magnetic tweezers or opticaltweezers. Optical tweezers have beenbroadly used to study practically allclasses of molecular motors as discussedin previous chapters. The notableexception is the helicases. Although anassisting force on substrate was used toinvestigate the unwinding of DNAduplexes (Dessinges et al. 2004;Dumont et al. 2006) no directmeasurements of forces exerted by ahelicase have been reported yet. In this

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study we present preliminarymeasurements of the force-velocityrelationship for P4 using opticaltweezers. In the optical tweezers adielectric particle, such as silicamicrosphere is attracted to and trappednear the waist of a laser beam that hasbeen focused through a high numericalaperture microscope objective (Ashkin1997). The position of the trapped beadcan be monitored with nanometer orbetter accuracy (Nugent-Glandorf andPerkins 2004). An applied external force

displaces the trapped bead from the trapcenter, with a linear dependence ofdisplacement on force. This allows themeasurement of force in the range of0.1-100 pN. (Visscher and Block 1998).Velocity versus force measurements atdifferent ATP concentrations allow forthe identification of the catalytic stepthat is coupled to force generation(Keller and Bustamante 2000;Bustamante et al. 2004).

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B. AIMS OF THE STUDY

When this work was initiated, onlylimited information was available on themechanism of action of the packagingmotor P4. It was known that isolated P4hexamers translocate along ssRNA inthe 5’ to 3’ direction and P4s frombacteriophages 8 and posseshelicase activity. While colleagues wereworking to solve the P4 structure thedetails of the P4 enzymology and themechanism of RNA translocationremained elusive.

The aim of this study was todetermine the mechanism of RNAtranslocation by viral packaging motorP4. Specifically:

- Identify steps (substrate binding,hydrolysis, product release) inthe enzymatic mechanism of P4.Estimate their rates and comparethese among the P4s fromdifferent Cystoviridae familymembers.

- Determine the order andcoordination of individualenzymatic and RNAtranslocation steps around the P4hexamer.

- Study P4 translocation alongRNA at the single moleculelevel.

- Study the dynamics of P4 duringRNA loading and translocationusing hydrogen-deuteriumexchange.

- Inspect changes of P4 dynamicswithin the viral procapsid andtheir possible implications for P4regulation.

- Integrate the experimental resultsin a physically feasible model ofRNA translocation and mechano-chemical coupling in P4

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C. MATERIAL AND METHODS

C.1. Reagents and preparations

Purified recombinant P4s and theirmutants were used in this work. Theplasmids which were used to express theproteins in E. coli cells are listed inTable 1. Nucleotide binding by P4 wasstudied using fluorescence spectroscopy.In the case of 8 P4 changes in spectraof fluorescent TNP-nucleotide analogueswere measured (Study I). In the case of

12 P4 fluorescence resonance energytransfer (FRET) between P4 tryptophansand MANT-nucleotide analogues wasmeasured (Study II). Nucleotidehydrolysis and subsequent phosphaterelease were detected spectroscopicallyusing EnzChek phosphate assay kit(Molecular Probes) (Study I, II, IV andV). Gel mobility shift assay was used to

measure P4-RNA complex formation(Study I and III). Standard gelelectrophoresis assays were used todetect RNA synthesis (Study V). Ramanspectroscopy was used to analyze P4complexes with nucleotides and RNA(Study I). HDX detected by highresolution (FT-ICR) mass spectrometrywas used to study changes in P4dynamics and structure upon interactionwith nucleotides and RNA (Study III)and with viral procapsid (Study IV).Tethered particle motion method andoptical trapping were used to measureRNA translocation by P4 at the singlemolecule level (Study V, unpublishedresults).

Table 1. Plasmids used in this studyName Description ReferencepSJ1b 8 P4 wt (Kainov et al. 2003b)pPG27 12 P4 wt (Mancini et al. 2004b)pDK21 8 P4 with deleted LKK 184-186 IIIpDK85 8 P4 with K185A mutation IIIpDK86 8 P4 with K186A mutation IIIpDK50 12 P4 with truncated C-terminus (I321Stop) IVpDK8P4H 8 P4 with C-terminal his-tag (Howorka, Kainov and

Tuma, manuscript inpreparation)

C.2. Data analysis and software tools

We have developed five softwarepackages to analyze and visualize datacollected by HDX and TPM methods:

Sequence Search is a program developedin Visual Basic. Its function is toidentify peptide-like patterns in massspectra and assign them by comparisonof the measured peptide monoisotopic

mass with the predicted protein fragmentmasses.Mass Search program (developed inVisual Basic) finds the peptide isotopicpattern of a given monoisotopic m/zvalue and charge in a measuredspectrum. It then calculates centroid ofthe pattern, pattern width and the

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centroid error (if more than oneindependent trial is available). If thepeptide assignment (i.e. number ofexchangeable hydrogens) and fullydeuterated mass are known then itcalculates also the deuterium content.Finally, it generates HDX kinetics frommultiple time-point spectra.

HDX Analysis program (developed inMatlab) allows plotting and comparisonof the measured HDX kinetics and thecorresponding exchange ratedistributions, which are Laplacetransformations of the kinetics(calculated using Maximum EntropyMethod (Zhang et al. 1997)). Itcalculates numbers of slow, fast, andintermediate rate exchanging hydrogens.It also generates sets of commands thatcan be used to color molecular modelsaccording to the HDX rates within theUCSF Chimera software (Huang et al.1996).Bead Tracing program (developed inMatlab) processes TPM video-microscopy records. It identifies allobjects in a given video frame usingintensity threshold detection method.Then it selects spherical objects of thecorrect size (i.e. the images of themicrospheres) and registers theirpositions within each frame (Fig. 12).

Bead Analysis program (developed inMatlab) shows graphically all recordedpositions of a selected bead, correctsthem for a stage drift, calculates theirRMSD as a function of time andhistograms the bead position along the Xand Y axes. This helps to distinguishbetween stuck, single-tethered andmultiple-tethered beads (Fig. 13). Thestuck and multiple-tethered beads areeliminated from further analysis.

Figure 12. Illustration of the imageanalysis by ‘Bead tracing’ program. (A)One frame of a video-microscopy sequenceshowing the 1 m silica beads tethered to amicroscope cover-slide via the P4-RNAcomplexes. (B) The objects that weredetected by the ‘Bead tracing’ program inthe original image (A). (C) The ‘bead-like’objects that passed through the size andshape filters.

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Figure 13. Typical patterns of the tethered bead jiggling observed in video-microscopysequences. (A) A bead which is stuck directly to the surface of microscope cover-slideexhibits practically no motion. (B) A bead which is tethered by several RNA moleculesexhibits a significant motion, however, the motion pattern is not circular. (C) A bead which istethered by single RNA strand exhibits a significant motion with a circular pattern.

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D. RESULTS AND DISCUSSION

D.1. NTP binding, hydrolysis,and NDP release

P4, like other molecular motors,works in a cyclic manner. P4 catalyticcycle is composed of NTP binding, NTPhydrolysis and product (NDP andphosphate) release. We have studied indetail the catalytic cycle of P4s frombacteriophage 8 (Study I) and 12(Study II). Among the family ofCystoviridae , 12 and 8 P4s wereselected for the following reasons: (i)

12 P4 has a known structure (Manciniet al. 2004a) which constitutes theframework for interpretation ofbiochemical results (ii) isolated 8 P4stranslocate RNA processively in vitro(Kainov et al. 2003b). Thus, themechanism of motor action can bestudied for 8 P4 and the results can beinterpreted in terms of 12 structure.

Several common features of thecatalytic cycle have been found for 8and 12 P4s (Study I and II). All sixbinding sites within the P4 hexamerappeared equivalent and no ATP bindingcooperativity was detected. RNAbinding stimulated ATP hydrolysiswhich was cooperative and had anapparent Michaelis constant close to theATP dissociation constant. On the otherhand, RNA had no effect on ATPaffinity or ADP release. ADP releasewas fast and independent of phosphaterelease. ADP binding had an equilibriumconstant close to that of ATP binding.This implies that the catalytic cycle isdriven by the difference between cellularconcentrations of ATP and ADPrespectively. This was further confirmedby the inhibition experiments (Study II).

To interpret the observedfeatures we proposed a “stochastic-sequential” cooperativity model (StudyII). In this model, the apparentcooperativity is a result of hydrolysis

stimulation by ATP and RNA binding toneighboring subunits rather thancooperative nucleotide binding.Simultaneous interaction of neighboringsubunits with RNA makes the otherwiserandom hydrolysis sequential andprocessive.

D.2. RNA binding

8 P4 forms a stable complexwith RNA (Study I). RNA binding to aprimary site on the perimeter of P4induces P4 ring opening which enablesslipping of RNA into the central channelof the hexamer. Thus, P4 encloses RNAtopologically (Study III). Similar ringopening during NA loading (Fig. 6) hasbeen demonstrated also for T7 gp4(Ahnert et al. 2000) and Rhotranscription terminator (Kim and Patel2001; Skordalakes and Berger 2003) andmight be a common feature of allhexameric helicases. Ring opening andclosing proceed in the absence as well asin the presence of nucleotides (StudyIII). However, the nucleotide binding byP4 might affect the kinetics of ringopening and/or closing and thus changethe apparent P4 affinity for RNA (StudyI).

The ring opening plays animportant role in the viral life cycle(Study III). The 5’-end of the viralgenome precursor ssRNA (containingrecognition pac sequence) is specificallyrecognized by the major capsid proteinP1 (Mindich 1999) and consequently nofree 5’ end is available for directthreading through P4. However, thespecific binding of the viral RNA to P1brings the RNA to the vicinity of P4 andthus triggers ring opening followed byRNA loading. After loading, thepresence of RNA in the central channelactivates the P4 motor resulting in

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processive RNA translocation into thevirion.

P4 interacts with the RNA sugar-phosphate backbone (Study I). Mostlikely, the negatively charged RNAphosphate groups electrostaticallyinteract with the positively charged tipof the P4 6 helix (Mancini et al.2004a). However, this interaction doesnot resemble the tight binding seen forspecific RNA-protein complexes.Instead, the relatively weak interactionsallow hindered diffusion along the RNAstrand, as determined by TPMexperiments (Study V). Such a one-dimensional diffusion is common forcertain NA binding proteins (Blainey etal. 2006; Graneli et al. 2006). In the caseof P4, the diffusion is hindered byactivation energy barriers that depend onthe nucleotide binding state (Study V).The estimated height of the activationbarrier implicates the formation of onesalt bridge between the P4 hexamer andRNA in the absence of nucleotides. Inthe presence of ATP or ADP, two saltbridges form, presumably, involving twoP4 subunits (Study V). Some additionalinteractions (e.g. hydrogen bondingbetween RNA and P1 loop of P4) may

be involved in the RNA binding.However, these are invariant to the P4nucleotide binding state (Study V).

D.3. RNA translocation

Ensemble measurements haveshown that P4 unwinds dsRNA in 5’ to3’ direction (Kainov et al. 2003b).Single molecule experiments (Study V)revealed that in the presence of ATP,unidirectional net movement towards theRNA 3’-terminus appeared in addition tothe diffusive motion which wasdiscussed above. Results of the singlemolecule experiments imply that P4employs an “electrostatic friction clutch”instead of cycling through stable,discrete, RNA binding states (Study V).ATP hydrolysis generates changes in theelectrostatic potential inside the centralchannel which in turn biases RNAmotion down the central channel (StudyV). Thus, P4 can be viewed as aBrownian motor that biases the randommovements in one direction (Astumian1997; Levin et al. 2005).

Figure 14 (A) Preliminary results of RNA translocation by P4 at saturating ATPconcentration measured by optical tweezers. (B) Translocation speed calculated from datashown in panel (A), and plotted as a function of applied force. The experimental setup wassimilar as in TPM measurements. Note that these are preliminary results obtained with anapproximate optical tweezers calibration. (Wallin, Lisal, and Tuma, unpublished data).

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The maximum translocation rateat the saturating ATP concentration wasvmax = 9.9 ± 0.2 b/s (Study V). The ATPhydrolysis rate is approximately 5 ATP/sat 20°C (Study I). Thus, the measuredrate of translocation corresponds to 2bases translocated per one ATPhydrolyzed. This is in perfect agreementwith the structural model of RNAtranslocation (Mancini et al. 2004a) andwith estimates for other hexamerichelicases (Kim et al. 2002). Preliminaryresults of experiments using an opticaltrap to detect P4 translocation yieldedthe same initial translocation speed of~10b/s at zero load and a stalling forceof ~6 pN (Fig. 14) (Wallin, Lisal, andTuma, unpublished data). The lineardecrease of P4 speed with increasingload (Fig. 14B) and the low stallingforce are consistent with the“electrostatic friction clutch” hypothesis.

D.4. Mechanism of singletranslocation step

Raman spectroscopy revealedthat RNA translocation is not associatedwith secondary structure changes in 8P4 (Study I). Thus, it must be associatedwith tertiary structure changes (e.g. themovement of 6 helix as observed in thecrystal structure of 12 P4 (Mancini etal. 2004a)) or with quaternary structurechanges (e.g. subunit rotation asobserved in the crystal structure of T7gp4 (Singleton et al. 2000)). Todistinguish between these two optionswe performed HDX measurements on 8P4 in the complex with differentnucleotides and RNA in solution (StudyIII). HDX kinetics reflects proteindynamics and surface solventaccessibility. HDX kinetics of thecatalytic RecA-like domain responded ina concerted manner to nucleotide andRNA binding while subunit interfaces

remained unaffected. These results ruledout quaternary structure changes duringRNA translocation (Study III).Therefore, HDX study confirmed thatthe mechanism based on structuralstudies of 12 P4, which involvestertiary structure changes within alimited protein region, applies also to 8P4. This suggests that the samemechanism might be universallyapplicable to P4s from otherbacteriophages as well as to otherhexameric helicases.

D.5. Mechanism of stepcoordination

We find that RNA interacts withseveral P4 subunits simultaneously(Study II and V). The P4-RNAinteraction turns the otherwise randomlyoccurring ATP hydrolysis into asequentially ordered one (Study II).RNA binding by neighboring subunitsfacilitates the transition state formation(Fig. 15B). This does not lead to achange in the nucleotide affinity butincreases the probability of ATPhydrolysis by neighboring subunits.RNA binding affinity is in turnmodulated by ATP binding (Study V)and by ATP hydrolysis (Study II).Indeed, both the ATP binding and theATP hydrolysis lead to conformationalchanges of the RNA binding site(Mancini et al. 2004a). The proposedmechanism of ATP hydrolysis and RNAtranslocation coordination isschematically depicted in Fig. 15.Although we originally proposed adifferent mechanism for 8 P4 (StudyI), the later data (Study III) rule out theproposed subunit movements andsuggest that 8 P4 employs a mechanismsimilar to that of 12 P4.

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Figure 15 Model of catalytic cooperativity for 12 P4. (A) Mechanism of ATP hydrolysisin the absence of RNA. Blue squares represent P4 subunits within the hexamer unraveled inplane. The red symbols designate binding sites occupied by ATP. The double-headed arrowsindicate independent stochastic binding to nucleotide sites. Lower panel shows the randomlyattained, “three in the row”, configuration that permits hydrolysis at the middle site (orange).Note that hydrolysis also requires the correct conformation of the key side chains fromneighboring subunits. Arrows depict the proposed communication between subunits. (B) ATPhydrolysis in the presence of RNA. Triangular appendages correspond to the RNA bindingL2 loop. Green symbols mark the ADP binding sites and pink symbols designate the bindingsites that are occupied by ATP at the high cellular ATP concentration but may be empty orbind AMP-PNP or ADP under defined laboratory conditions. The yellow line and circlesrepresent the RNA sugar phosphate backbone. Arrows depict the proposed communicationbetween subunits. For details of the proposed mechanism see Study II.

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D.6. P4 activity regulation withinthe polymerase complex

P4 catalytic activity is regulatedin the context of the polymerase complex(Kainov et al. 2004). The ATPase isactive during packaging but shuts off fortranscription. The mechanism of P4activity regulation by the polymerasecomplex was elusive. Our HDX studiesrevealed that P4 associates with theprocapsid (PC) via its C-terminal facet(Study IV). Association of the C-terminus with the PC affects thedynamics of the structurally-contiguouscatalytic RecA-like domain (Fig. 16) andmay affect ATPase activity. Theinfluence of the C-terminus on theATPase was examined by use of a P4 C-

terminal deletion mutant (Study IV).Together, the results show that the C-terminus acts as an inhibitor of ATPaseactivity in the isolated P4 hexamer. Wepropose that in the virus core the C-terminal helix may be either withdrawnfrom the hexamer to activate the ATPase(procapsid in the packaging state) orjammed onto the catalytic RecA-likedomain to shut off the activity(polymerase complex in transcriptionmode).

The interaction with the PC alsoprevents the spontaneous P4 ringopenings and thus heterologous RNAbinding (Study IV). The PC likelytriggers ring openings in a controlledfashion in order to selectively load theviral RNA during packaging initiation.

Figure 16 12 P4 subunit colored according to the HDX rates for isolated P4 hexamer(A) and the hexamer within the procapsid (B). The subunit is drawn in the sameorientation as in Fig. 10. The central channel of P4 is in the front, the C-terminal base is atthe bottom, the N-terminal apical domain points up. The scale bar for HDX rates (h-1) isshown at the bottom.

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D.7. Implications for othersystems

The mechanism that we proposefor P4 combines features of bothhexameric helicase models that werediscussed in chapter A.2.2. It assumesthe sequential order of ATP hydrolysisby P4 subunits similarly as the T7 gp4model, and it employs tertiary structurechanges within a limited subunit region,similarly as the SV40 LTA model. Wepropose that the P4 mechanism might beuniversally applicable to all hexamerichelicases including both T7 gp4 andSV40 LTA. We suggest that theobserved rotations of T7 gp4 subunits(Singleton 2000) might be related to thering opening during DNA loading(Ahnert 2000) rather than to DNAtranslocation mechanism similarly as weobserved for P4 (Study III). Further, wesuggest that the concerted hydrolysismodel proposed for SV40 LTA (Gai etal. 2004) might be a result of incorrectinterpretation of the structures that wereobtained under non-physiologicalconditions (no DNA; ATP or ADPonly). Indeed, a recent structure of therelated E1 helicase in a complex withDNA (Enemark and Joshua-Tor 2006)exhibits a spiral staircase arrangement ofthe DNA binding loops, which is verysimilar to that proposed for P4 (Fig. 15).

Mechanism similar to that of P4may also apply to other AAA+ motors.However, the details of the mechanismmay differ for individual AAA+ motors

as they are optimized for their specificfunctions. For example, the sequentiallyordered hydrolysis will emerge onlywhen there is an advantage to it - i.e.when the translocation substrate has aproper kind of periodicity (like a NA).When the translocation substrate isasymmetric or random (like anunstructured polypeptide), stochasticATP hydrolysis by individual subunitsmay be sufficient and robust to achieveprocessive translocation (Martin et al.2005).

Another frequently discussedissue is the rotation of P4 and/or RNAwith respect to the procapsid duringpackaging in a fashion proposed for 29packaging motor (Simpson et al. 2000).In our model, no rotation of P4 relativeto the procapsid or RNA relative to P4 isrequired. In fact, a stable position of allparts involved in packaging is necessaryto keep the correct alignment of RNAphosphate groups with respect to P4subunits which promotes sequentialhydrolysis and processive RNAtranslocation. In addition, recent cryo-EM studies of the asymmetricpolymerase complex vertices (includingP4) show that P4 rotation relative to thepolymerase complex core during RNApackaging is unlikely (Huiskonen et al.2006). The role of the symmetrymismatch between the procapsid five-fold vertex and the P4 hexamer might beto allow for ring opening during RNAloading (Study III).

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E. CONCLUSIONS AND FUTURE PROSPECTS

We studied the viral ssRNApackaging motor P4 using bothensemble and single-moleculetechniques. We focused on itsenzymology, interactions with RNA,translocation along RNA, andinteractions with the viral procapsid. Weintroduced the hydrogen/deuteriumexchange method to study molecularmotors. We delineated the principles ofP4 mechanism and discussed theirimplications for other hexamericmolecular motors.

In order to unravel further detailsof the P4 mechanism the solution of itsstructure in complex with RNA will benecessary. According to our conclusionsRNA translocation requires asymmetryin the occupancy of P4 binding sites by

nucleotides. Thus, successfulcrystallization of P4-RNA complex willneed co-crystallization of P4-RNA withdifferent mixtures of ATP/ADPanalogues. Details of P4-RNAinteractions could be also deduced fromsingle molecule studies of the diffusionand active translocation of selected P4mutants along different RNA substrates.

A fairly good understanding ofthe P4 mechanism should make itpossible to use P4 in a range ofbionanotechnological applications in thenear future. P4 may act as a RNA(DNA) pump in nanodevices which willbe used, for example, for sequencing ofsingle RNA (DNA) molecules(Howorka, Kainov and Tuma,manuscript in preparation).

36

F. ACKNOWLEDGEMENTS

My deepest gratitude goes to mysupervisor Doc. Roman Tuma for hisexcellent ideas, support and always opendoor for discussions of all kinds. I wouldlike to thank all members of the FinnishCentre of Excellence in Virus Researchwho provided not only a professionalscientific but also personal atmospherethat made working in the lab a pleasure.Especially, I would like to thank Prof.Dennis Bamford, Sarah Butcher, AymanAbu Ramadan, Anders Wallin, DenisKainov, Tomas Malinauskas, MichaelMerckel, Juha Huiskonen, HarriJäälinoja, Pasi Laurinmäki, Nelli Karhu,Riitta Tarkiainen, Sampo Vehma andPetri Papponen.

I am grateful to my collaboratorsTuKiet Lam, Mark Emmett and Prof.Alan Marshall from the National HighMagnetic Field Laboratory at FloridaState University in Tallahassee, USA.

I deeply appreciate Prof. CarlGahmberg, head of the Division of

Biochemistry for his interest in mywork. I gladly acknowledge the supportfrom Viikki Graduate School inBiosciences and Prof. Marja Makarowand Dr. Eeva Sievi in particular. Further,I would like to thank members of myfollow-up group Prof. Pekka Hänninenand Prof. Michael Verkhovsky for theirgood advises. I thank the reviewers Prof.Lloyd Ruddock and Prof. Nynke Dekkerfor critical reading of the manuscript andconstructive comments.

I warmly thank all my friends fortheir support. Especially, I would like tothank the whole bunch of Czechspeaking people living in Finland andorganized in Fin ech club(http://fincech.wz.cz) for making mydays in Finland much easier and evenmore enjoyable. I would like to thankmy parents Ji í Lísal and R ženaLísalová and my sisters Veronika andAneta for their support and love.

37

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