Dynamic Behavior of the Telomerase RNA Hairpin Structure and its Relationship to Dyskeratosis...

doi:10.1016/j.jmb.2005.02.015 J. Mol. Biol. (2005) 348, 27–42

Dynamic Behavior of the Telomerase RNA HairpinStructure and its Relationship to DyskeratosisCongenita

Yaroslava G. Yingling and Bruce A. Shapiro*

Laboratory of Experimental andComputational Biology, NCICenter for Cancer ResearchNCI-Frederick, NationalInstitutes of Health, Building469, Room 150, FrederickMD 21702, USA

0022-2836/$ - see front matter Published

Abbreviations used: DKC, dyskermolecular dynamics; GB, Generalizmean-square deviation.

E-mail address of the [email protected]

In this paper, we present the results from a comprehensive study ofnanosecond-scale implicit and explicit solvent molecular dynamicssimulations of the wild-type telomerase RNA hairpin. The effects ofvarious mutations on telomerase RNA dynamics are also investigated.Overall, we found that the human telomerase hairpin is a very flexiblemolecule. In particular, periodically the molecule exhibits dramaticstructural fluctuations represented by the opening and closing of a non-canonical base-pair region. These structural deviations correspond tosignificant disruptions of the direct hydrogen bonding network in the helix,widening of the major groove of the hairpin structure, and causing severalU and C nucleotides to protrude into the major groove from the helixpermitting them to hydrogen bond with, for example, the P3 domain of thetelomerase RNA. We suggest that these structural fluctuations expose anucleation point for pseudoknot formation. We also found that mutationsin the pentaloop and non-canonical region stabilize the hairpin. Moreover,our results show that the hairpin with dyskeratosis congenita mutationsis more stable and less flexible than the wild-type hairpin due to basestacking in the pentaloop. The results from our molecular dynamicssimulations are in agreement with experimental observations. In addition,they suggest a possible mechanism for pseudoknot formation based on thedynamics of the hairpin structure and also may explain the mutationalaspects of dyskeratosis congenita.

Published by Elsevier Ltd.

Keywords: telomerase; RNA; molecular dynamics simulations; dyskeratosiscongenita; pseudoknot
*Corresponding author
Introduction

Telomeres are DNA sequences located at the endsof eukaryotic chromosomes and contain thousandsof TTAGGG sequence repeats.1–3 In most normalcells, telomeres are shortened at each cell divisionuntil a critical length is reached prohibiting furthercell division. This mechanism is vital for maintain-ing limited proliferative capacity in normal cells.Maintenance of a minimal telomeric DNA length isa necessity for chromosome stability and preven-tion of oncogenic mutations. The elongation oftelomeres is performed by the ribonucleoprotein

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atosis congenita; MD,ed Born; RMSD, root-

ing author:

telomerase. It replicates telomeres using its RNAtemplate and therefore prevents telomeric DNAfrom being shortened. Thus active telomeraseprevents erosion of the chromosome ends andsupports unlimited cell divisions. Telomerase isclosely related to cellular immortality and is activein most malignant human tumors and cancerouscells.4 On the contrary, it is repressed in mostnormal somatic adult tissues, however, it is active instem cells, germ line cells, inflammatory cells, andcells in other self-renewing tissues.5 The ability toregulate cellular activity makes telomerase a poten-tial target for cancer development, progression,diagnosis, clinical outcome prediction, and cancer-specific targeting treatment and prevention.6–10

The main components of telomerase are itsreverse transcriptase and telomerase RNA contain-ing a short template sequence (Figure 1(a)). Thestability of the ribonucleoprotein complex andtelomerase activity require the formation of a

Figure 1. (a) Secondary structure of the highly conserved template/pseudoknot domain of human telomerase RNA,which consists of the template region, the pseudoknot domain, and the P1b helix. The secondary structure is based onthe phylogenetic analysis of the vertebrate telomerase RNA and includes base-paired regions that are universallyconserved in all vertebrates.16 The shaded box represents the RNA sequence used in this study. The nucleotides involvedin the DKC mutation are circled. The secondary structures of the RNA sequences used in this study: (b) the wild-typehairpin structure; (c) the DKC-mutated hairpin structure; (d) the test hairpin structure. The modified residues arecolored in red. (e) Lowest energy structure of the wild-type hairpin structure. The U bases are blue, the C bases are red,the A bases are green, and the G bases are purple.

28 Dynamic Behavior of Telomerase RNA

pseudoknot in telomerase RNA.11 This pseudoknotis a dynamic structure as suggested by chemicaland enzymatic probing11 and can exist in twoalternative stable states in solution.12 The firststate consists of a hairpin pentaloop domain aloneand the second state contains a pseudoknot formedby pairing the P3 domain with the hairpinpentaloop domain. The equilibrium between thesetwo states controls the optimal telomerase function-ing.12 Moreover, recent in vivo and in vitro structuralanalyses of telomerase RNA suggest that theevolutionarily conserved and functionally essen-tial pseudoknot is formed only temporarily intelomerase and that dynamic conformational

rearrangement between these two states is essentialfor telomerase functioning and acts as a biologicalswitch.13

Mutations within telomerase can dramaticallyaffect enzyme activity, fidelity, and processivity oftelomerase.3 Specific mutations impair the haemo-poietic function of telomerase in humans and havebeen associated with inherited bone marrow failureas in aplastic anaemia and dyskeratosis congenita(DKC). Some forms of DKC are caused bymutations in telomerase RNA, which consequentlyreduce telomerase activity and thus produce veryshort telomeres.14 DKC mutations of telomeraseRNA are important for telomerase catalysis and

Dynamic Behavior of Telomerase RNA 29

RNA stability. Moreover, the specific mutations intelomerase disrupt the base-pairing associated withthe P3 domain of the pseudoknot and preventthe stable assembly with the catalytic reversetranscriptase component of telomerase11 resultingin deleterious cellular growth, telomere main-tenance, and the reduction of telomerase activity15

by as much as 50% causing haploinsufficiency.16

To understand the effect of the correct folding ofthe pseudoknot on telomerase activity and hencecellular proliferation, the DKC mutation, whichinvolves two base substitutions in the telomeraseRNA, has been examined by using in vitro telomer-ase assays, NMR, and UV absorbance meltinganalysis.12 The results have shown increasedstability of the hairpin pentaloop structure and asimultaneous decrease in the number of potentialbase-pairs in the pseudoknot, which, therefore,alters the functionality of telomerase. The effectsof the DKC mutations on the structure andthermodynamic stability of human telomeraseRNA have been investigated by using NMR andthermal melting.17,18 These experimental obser-vations found that the wild-type pseudoknotdominates the hairpin structure by approximately95%, while the DKC mutation leads to a shift inequilibrium to a 50/50 conformation. This obser-vation is supported by free energy calculations.19

Although an increasing number of telomerase-associated studies have been conducted, the under-standing of the specific role of the pseudoknotdomain is not yet established. Understanding thevarious functions and dynamic structural changesof telomerase may result in a clearer knowledge ofits multiple effects on cellular activity and telomeremaintenance, DKC disorder, aging, and cancer.Despite active ongoing investigations little isknown about the mechanism of formation of thepseudoknot or the dynamical changes taking placein the human telomerase hairpin structure priorto the formation of the pseudoknot. Another

interesting aspect is the effect of mutations on thedynamical characteristics of a telomerase RNA.

The dynamic analysis of three-dimensional struc-tures can provide key information to the under-standing of the RNA’s activity and structure–function relationships. Current experimental tech-niques are not capable of generating a completedescription of the dynamical structural behavior ofnucleic acids. Molecular dynamics (MD) simu-lations have produced reliable information onstructural changes and molecular motion of variousproteins and nucleic acids and have providedhighly detailed information not accessible by othermethods.20–31 Moreover, the rapid advances inparallel computing technologies and algorithmicmethodologies allow performing MD simulationson larger structures for longer periods of time.Despite the use of approximations and relativelyshort timescales, the results from MD calculationscomplement experimental observations and pro-vide valuable insights on structural and dynamicalchanges of various sizes of nucleic acids ranging,for instance, from a few nucleotides21 to a 75-nucleotide tRNA,31 a frame-shifting pseudoknot,25

and a 16 S rRNA–S15 complex.23

The goal of this paper is to present an accurateand comprehensive examination of the temporalstructural rearrangement of a segment of thetelomerase RNA and to assess the effect ofmutations on the telomerase RNA hairpin. Wehave employed molecular dynamics simulations toprobe the structural and dynamical changes of thetelomerase RNA. By performing our calculationsover nanosecond timescales we can access compre-hensive information and detect conformationalchanges in the RNA molecules. The simulationswere predominantly performed using a General-ized Born (GB) implicit solvation energy method.However, to make sure that the implicit solventmethod did not alter hairpin structure dynamics wealso performed a 20 ns explicit solvent simulation

Figure 2. Temporal RMSD of(a) the wild-type hairpin structure,(b) the test hairpin structure, and(c) the DKC-mutated hairpinstructure using GB method.Letters A, B, C, D, E, and F denotethe approximate locations of theflips in the trajectory.

Figure 3 (legend opposite)



using Particle Mesh Ewald (PME) summation on awild-type human telomerase hairpin structure. Ourresults indicate that the wild-type telomerase hair-pin structure undergoes large periodic structuraldeviations, which may possibly generate a nuclea-tion point for pseudoknot formation. Mutations inthe pentaloop and in the non-canonical region,however, stabilize the structure. The results may beof general importance considering that the telomer-ase RNA hairpin sequence with the four consecu-tive non-canonical base-pairs is conserved in mostvertebrate telomerases, including mammals, birds,amphibians, fish, and reptiles.32

Results and discussion

The time dependence of the root-mean-squaredeviation (RMSD) from the average structure forthe wild-type telomerase hairpin structure (Figure1(b)) is presented in Figure 2(a) over a 20 ns GBtrajectory. The RMSD characterizes this hairpin as astable and highly flexible structure. Moreover, thereare distinct large periodic fluctuations or flips of theRMSD at 3.9 ns (A), 7.5 ns (B), 10.8 ns (C), 16 ns (D),and 19 ns (E). These flips indicate that thereare important and dramatic structural changesoccurring in the telomerase RNA hairpin structure.A detailed examination of the structure at theseflips reveals critical changes in hydrogen bondingand the major groove of the hairpin structure. Forexample, the lowest energy wild-type hairpinstructure (Figure 1(e)) is strikingly different fromthe structure that occurred at the flip around 3.9 ns(Figure 3(a)). From this snapshot we can see that atthe flip several base-pairs are no longer aligned forhydrogen bonding, and U114, U113, and C112 basesare protruding from the helix into the major groove.To highlight the areas of structural divergence at theflip the backbone atoms of the most stable region(residues 93–98 and 116–121) in the lowest energywild-type hairpin structure and the structure at the3.9 ns flip were superimposed (Figure 3(c)). Thesuperimposed region near the terminal endsremains unchanged at the flip with an RMSD ofabout 1 A. However, the dramatic modificationsof the backbone positions near the non-canonicalregion and the pentaloop are evident.

The stability of an RNA molecule depends onbase-pairing (hydrogen-bonding) and base-stack-ing interactions. We first examine the hydrogen-bonding network and then the base-staking

Figure 3. (a) Stereo snapshots of the wild-type telomeraseblue, the C bases are red, the A bases are green, and the G baprotrusion of C112 and U113 into the helix can be seen. (b) Ster(point A). This represents the opening of the base-pairs a(c) Backbone representation of the lowest energy wild-type h(red). Superposition was performed on the backbone atorepresentations of the base-pairing occurring in the hairpin hthe U:U 0 base-pair, and (g) second conformation of the U:U 0 babonds between the bases.

interactions in an effort to discover the reason forthe structural fluctuations of the telomerase RNAhairpin.

Base-pairing in telomerase

The two polynucleotide chains in a helix areconnected by hydrogen bonds between comple-mentary bases. Only isomorphous Watson–CrickC:G and A:U base-pairs lead to a regular double-helical structure, whereas mismatch pairs, such asU:U and U:C, generally destabilize a structure. Forexample, the interaction energy in a vacuum, ascalculated by quantum chemical methods, for a G:Cbase-pair is K16.79 kcal/mol, A:U is K7.00 kcal/mol, and U:U is K5.42 kcal/mol.33 The humantelomerase RNA hairpin structure includes fourconsecutive conserved non-canonical base-pairsin a continuous 12 base-pair helix capped by apentaloop.17 The instability of the non-canonicalregion in the wild-type telomerase hairpin structureis more visible in Figure 3(b) where the regionbetween the U99:U115 and the U103:A111 base-pairs is depicted at the flip. Clearly, there is asignificant disruption in the direct hydrogen bond-ing: the U99:U115 base-pair has two direct hydro-gen bonds, U100:U114 has one hydrogen bond, andU101:U113, U102:C112, and U103:A111 are open;therefore, there is only one base-pair, C104:G110,separating this unstable region from the pentaloop.Moreover, the U114, U113, and most vividly C112nucleotides are rotated outward from the helix,which makes them accessible for bonding with theP3 domain.

In order to analyze the dynamical/structuralfluctuations of the base-pairs in this non-standardregion we have examined the time course of thehydrogen bond distances. In Figure 4 the directhydrogen bond distances for U99:U115-U103:A111are displayed and Table 1 represents the hydrogenbond occupancy for the region. There are twoalternatives for the U:U base-pairs in an asymmetricenvironment, hydrogen bonds between O2(U)/N3(U 0) and N3(U)/O4(U 0) as in the first confor-mation (Figure 3(f)) or hydrogen bonds betweenO4(U)/N3(U 0) and N3(U)/O2(U 0) as in the secondconformation (Figure 3(g)).33 All three U:U base-pairs in the wild-type hairpin structure can adoptboth conformations and switch sporadicallybetween these conformations throughout the tra-jectory (Figure 4(c)–(h)). For example, the U99:U115and U100:U114 base-pairs are in the second

RNA hairpin at the 3.9 ns flip (point A). The U bases areses are purple. The widening of the major groove and theeo view of the U99:U115-U103:A111 region at the 3.9 ns flipnd the rotation of C112 onto the side of the structure.airpin structure (blue) and the structure at the 3.9 ns flipms of residues 93–98 and 116–121. (d)–(g) Structuralelix; (d) A:U, (e) C:U, (f) first asymmetric conformation ofse-pair. The dotted red lines represent potential hydrogen

Figure 4. Time dependence of hydrogen bond distances for (a) H61(A111)/O4(U103) (red) and H3(U103)/N1(A111)(black); (b) H3(U102)/N3(C112) (black) and H41(C112)/O4(U102) (red); (c)H3(U101)/O2(U113) (black) andH3(U113)/O4(U101)(red); (d) H3(U113)/O2(U101) (black) and H3(U101)/O4(U113) (red); (e) H3(U100)/O2(U114)(black) and H3(U114)/O4(U100) (red); (f) H3(U114)/O2(U100) (black) and H3(U100)/O4(U114) (red);(g) H3(U99)/O2(U115) (black) and H3(U115)/O4(U99) (red); (h) H3(U115)/O2(U99) (black) andH3(U105)/O4(U99) (red) for the wild-type hairpin structure calculated using the GB method. The bold letters (A, B,C, D, and E) represent the approximate positions of the structural flip points. Atoms are considered to be hydrogen-bonded when the bond distance is less than 3.5 A.


conformation for w80% of the trajectory, whereasthe U101:U113 base-pair is predominantly in thefirst conformation. However, the transitionsbetween the two conformations are relativelyindependent of the flips. Yet there are short periodsof time when the base-pairs are open (at the flips)or there is only a single hydrogen bond betweenthe base-pairs. For example, a single or doublehydrogen-bonded base-pair exists between U99and U115 in one conformation or the other for92% of the trajectory, while for w60% of the timeonly one hydrogen bond is holding this base-pairtogether. The U100:U114 and U101:U113 base-pairsare held together by one (w20%) or two directhydrogen bonds (w70%) most of the time. Theunusual bonding between UU bases in the helixwith the two UU mismatches and one GU wobblepair has been previously observed and discussed.34

The observations show that UU base-pairs can

be found in both conformations in crystal struc-tures, where one UU pair is highly twisted and hasonly one hydrogen bond; therefore, exhibitingsimilar structural changes as we observed in oursimulations.

Next in the helix is the UC mismatch pair. UCbase-pairs usually contain only one direct hydrogenbond between O4(U) and N4(C) (Figure 3(e)),however, a second hydrogen bond is possiblebetween the two N3 nitrogen atoms presumablybridged by a water molecule. The U102:C112 base-pair is the least stable base-pair in the whole wild-type hairpin structure with an 87% N4(C112)/O4(U102) hydrogen bond occupancy (Figure 4(b)).The U103:A111 base-pair (Figure 3(d)) exhibits shortbase-pair opening events switching from two directbonds (60%) to one (28%) or to none. At the flipsthese bases have no direct hydrogen bonds (Figure4(a)). The last base-pair that separates this unstable

Table 1. Hydrogen bond occupancy

Base-pair Hydrogen bond Occupancy (%)

Wild-typehairpin, GB

DKC-mutatedhairpin, GB

Wild-typehairpin, PME

G98:C116

H41(C116)/O6(G98) 91.25 93.13 98.46H1(G98)/N3(C116) 97.12 98.41 99.61H21(G98)/O2(C116) 99.55 99.73 99.98

U99:U115I

H3(U115)/O2(U99) 22.36 61.68 99.21H3(U99)/O4(U115) 83.12 62.48 99.47

IIH3(U99)/O2(U115) 6.89 25.95 0.00H3(U115)/O4(U99) 9.37 30.11 0.00

U100:U114I

H3(U100)/O4(U114) 61.38 12.12 0.00H3(U114)/O2(U100) 77.85 11.34 0.00

IIH3(U114)/O4(U100) 12.56 62.77 72.65H3(U100)/O2(U114) 17.99 78.86 99.61

U101:U113I

H3(U101)/O4(U113) 9.61 55.68 57.57H3(U113)/O2(U101) 14.74 70.93 68.64

IIH3(U113)/O4(U101) 59.37 20.72 29.73H3(U101)/O2(U113) 81.64 25.13 30.28

U102:C112H3(U102)/N3(C112) 77.69 84.88 81.91H41(C112)/O4(U102) 86.79 88.60 83.14

U103:A111H61(A111)/O4(U103) 59.23 90.06 88.46H3(U103)/N1(A111) 87.58 97.61 97.45

C104:G110

H41(C104)/O6(G110) 90.95 92.97 99.24H1(G110)/N3(C104) 93.80 97.72 99.80H21(G110)/O2(C104) 93.25 98.62 99.98

Hydrogen bond occupancy in percentage computed over 20 ns for the wild-type hairpin structure calculated using the GB method,DKC-mutated hairpin structure calculated using the GB method, and the wild-type hairpin structure calculated using the PME method.The maximum allowable bond length is 3.5 A. For UU base-pairs, I and II indicate Figure 8(c) and Figure 8(d) conformations,respectively, in the asymmetrical environment.


region from the pentaloop is C104:G110. This base-pair is relatively stable, however, it too has veryshort periods with no hydrogen bonds (6%)primarily at some flips. Therefore, the flips exhibitdramatic disruptions of the hydrogen bond net-work leaving the structure open for binding with,for example, the P3 domain in the telomerase RNAto form a pseudoknot.

Since the UC base-pair can be mediated by awater molecule we performed a PME explicit waterand ions molecular dynamics simulation on thesame wild-type hairpin structure for a period of20 ns. To determine if the same results can beobtained, we analyzed the PME calculations andobserved similar dynamics except that only oneextended flip occurred at 14.5 ns with the samestructural rearrangements as compared to severalshorter flips when using implicit solvent. Theagreement can be seen in Table 1 where hydrogenbond occupancy is calculated for GB and PMEsimulations. Even in explicit water simulations theU:C base-pair is still the least stable pair in the helixwith a direct hydrogen bond occupancy of around84%, which is less than the occupancy seen in theGB simulations by 4%. This is not surprising, sincethis type of flexibility of the water-mediated U:Cbase-pair has been observed in other moleculardynamics studies.21 The U101:U113 base-pairexhibits similar conformational changes to theother U:U base-pairs and is bonded by one or twodirect hydrogen bonds with periodic very shortbase-pair opening events. Also the U103:A111 is

bonded by one, two or no hydrogen bonds similarto observations in the GB simulations. Even thoughwe observed a single flip in the 20 ns time period forexplicit solvent calculations, the dynamical changesare similar to those observed in the GB simulationsbased on the hydrogen bond occupancy. The flip isillustrated in Figure 5, where the temporal changesof the hydrogen bond distances for U102:C112 andU103:A111 base-pairs are displayed for the PMEcalculations.

In order to investigate the impact of the flips onthe geometry of the non-canonical base-pairs wemonitored C1 0–C1 0 atom distances of the non-standard base-pairs (Figure 6). The fluctuations ofthese distances for all base-pairs at the flips aredistinct. Interestingly, the average distance betweenthe C1 0–C1 0 atoms in the UU base-pairs decrease byabout 1 A right before the switch from oneconformation to the other. In addition, when theUU base-pairs are held together by only onehydrogen bond the average distance increases byapproximately 0.8 A.

Consecutive non-canonical base-pairs have aslightly different distance than canonical base-pairs. For instance, the standard C1 0–C1 0 distancebetween Watson–Crick base-pairs is 10.7 A for GCand 10.4 A for AU, whereas the distance for UU is9.9 A and for water-mediated UC is 11.7 A.35 Thelarger distance for the UC base-pair represents thefact that the water-mediated bridge increases thedistance between the bases. The average distancesbetween C1 0–C1 0 atoms in the helix obtained from

Figure 5. Time dependence of hydrogen bond distances for (a) H61(A111)/O4(U103) (red) and H3(U103)/N1(A111)(black); (b) H3(U102)/N3(C112) (black) and H41(C112)/O4(U102) (red); for the wild-type hairpin structure calculatedusing the PME method. G represents approximate position of the long structural flip.


our simulations of the wild-type hairpin structureare presented in Table 2. Indeed, the distancesbetween non-canonical base-pairs are smaller thanbetween Watson–Crick base-pairs. However, thedistance between the UC base-pair is smaller thanthe requirement for a water-mediated bridge and issimilar in both simulations using GB implicitsolvent (8.96 A) and PME explicit solvent (8.83 A)calculations. Moreover, the distance between thefirst U99:U115 base-pair is larger than the distancebetween the other UU base-pairs inside the non-standard region. Albeit the middle UU exhibitsmore flexibility and regular conformational switch-ing, it is unknown whether the smaller or largerdistance plays an important role in the base-pairdynamics.

Furthermore, four consecutive non-standardbase-pairs have been found to influence the majorgroove width. For example, the experimentalinvestigation of the r-GGACUUCGGUCCsequence, which forms a duplex with an internalloop consisting of four consecutive U-G, U-C, C-U,and G-U mismatches, has shown that the major

groove width is almost doubled, to 7.5 A, at theinternal loop, suggesting a potential protein recog-nition site.36 Our molecular dynamics simulationsalso demonstrate that the major groove significantlyexpands at the flips. The major groove width istypically determined by the shortest P–P distancebetween the two strands of the nucleic acid, minus5.8 A for the radii of the two P atoms. The shortestP–P distance for the major groove width in ourstructure can be approximately measured from a Pi

atom on one strand to the PiK6 atom on the otherstrand. For the minor groove, the distance ismeasured between Pi and PiC3 atoms on theopposite strands. Our calculations indicate thatthe major groove of the wild-type hairpin structurevaries from 10 A to 24 A and fluctuates around itsaverage width of 15.8 A. At the flips the majorgroove width increases by 5 to 9 A. The averageminor groove width is around 6.5 A and oscillatesG2 A at the flips. We measured the value of theminor and major groove width for an A-form RNAhelix with canonical base-pairs and obtained 11.4 Aand 4.2 A, respectively. For a canonical B-form

Figure 6. Time course of C1 0–C1 0

distances between (a) U102:C112,(b) U101:U113, (c) U100:U114, and(d) U99:U115 base-pairs of thewild-type hairpin structure. Thebold letters represent the approxi-mate locations of the flips.

Table 2. Average distances between C1 0–C1 0 atoms in ahelix (A)

Base-pairsWild-type

hairpin, GBDKC-mutated

hairpin, GBWild-type

hairpin, PME

U97:A117 10.77 10.84 10.72G98:C116 10.61 10.60 10.58U99:U115 9.66 8.98 8.77U100:U114 8.54 8.44 8.27U101:U113 8.38 8.47 8.38U102:C112 8.96 8.91 8.83U103:A111 10.23 10.61 10.36C104:G110 10.88 10.63 10.68

GB indicates implicit solvation method and PME indicatesexplicit solvation method used for calculations


DNA the minor and major groove widths are 5.7 Aand 11.7 A respectively.33 Therefore, the majorgroove of the wild-type hairpin structure at theflip is even larger than that of B-DNA allowing forthe accessibility of another polynucleotide chain.

In order to investigate the importance of con-secutive non-standard base-pairs on the dynamicalfeatures of the telomerase RNA we also examine atest hairpin structure where the non-canonical base-pairs were replaced by the Watson–Crick base-pairs(Figure 1(d)) for a 10 ns time period. The time-dependent RMSD (Figure 2(b)) of the hairpinstructure is very stable. It fluctuates around 1.5 Aand exhibits no severe structural modifications.Therefore, the dynamical rearrangements of thethree consecutive U:U and one U:C base-pairsgreatly influence the overall dynamical charac-teristics of the hairpin structure.

Base-stacking interactions

The movements of the RNA are directly related tothe backbone torsion angles. Moreover, since thebases are linked to the backbone of the RNA, onemight suspect that the switching between thedifferent conformations of the UU base-pairs isdue to changes in the torsion angles of the back-bone. We performed a detailed analysis of thebackbone torsion angles of the wild-type hairpinstructure over a 20 ns trajectory and summarizedthem in Table 3. The torsion angles are definedaround their central bond and include a (P–O5 0),b (O5 0–C5 0), g (C5 0–C4 0), d (C4 0–C3 0), 3 (C3 0–O3 0),z (O3 0–P), and the glycosidic torsion angle c(C1 0–N). The maintenance of base-stacking inter-actions is reliant on angle c, which defines theorientation of the aromatic base with respect tothe sugar. Angle c is sterically restricted to eitherthe anti conformation (around K1808), which isstandard for A-RNA molecules, or syn confor-mation (around 608).33 Since angle c can beresponsible for the stability of RNA molecules weclosely examine the residues that have an unusual cangle. From the Table 3 we can see that residuesU105, C106, G107, U109, and U115 exhibit non-standard c angles for A-RNA. However, the

nucleotides from the non-canonical region displaystandard torsion angles, except for the U115nucleotide, which has a single change in its torsionangles at around 7.5 ns, when it switches from itstwo hydrogen bond conformation to its one hydro-gen bond conformation. The relative stability ofthe torsion angles of the UU bases leads to theconclusion that the conformational switches of theUU base-pairs are not the initiators of the periodicstructural changes in the wild-type RNA hairpinstructure.

Other bases with the unusual c angles are locatedin the pentaloop. Base G107, which is located at thetop of the pentaloop, has a stable c angle in the synconformation. In the syn conformation the bulk ofthe base is positioned over the sugar, giving rise toclose interatomic interactions, which can berelieved if the sugar adopts a C2 0-endo pucker,which is the case for G107. Rotation about theexocyclic C4 0–C5 0 bond (angle g) plays a crucial rolein the positioning of the phosphate group relativeto the sugar and the base in the nucleotides. Thepossible conformations of angle g are (C) gauche,(K) gauche, or trans. In purine nucleotides Ggaucheare standard g angle ranges, where trans is rarelyobserved only with C2 0-endo. In pyrimidine nucleo-tides, the preferred conformation is Cgauche (w608)regardless of the sugar pucker, with a few casesfound exhibiting Kgauche (wK608) and trans(w1808) with C2 0-endo.33 If the c angle is in synand g angle adopts a trans conformation as withG107 then a C–H/O5 0 bond can be formed,creating a “rigid nucleotide”.33 Our analysis of thetrajectory indeed demonstrates that G107 is the leastflexible base in the whole molecule. U105, C106, andU109 have dynamic c angles intermittently switch-ing from syn to anti conformations throughoutthe trajectory. Subsequently any of these residuescan provide movement destabilizing the RNAmolecule. The standard values for the torsion anglesa and g correspond to Kgauche and Cgaucheconformations. However, when the g torsion angleis in the trans conformation and a fluctuates over arange of angles, the distance between the O2 0 atomof this nucleotide and the O5 0 atom of the nextnucleotide becomes relatively short, permitting theformation of a hydrogen bond. As shown by theexplicit solvent molecular dynamics simulationsthese interactions may affect the backbone rigidityand the thermodynamic stability of the RNA helicalstructures.27 The residue that exhibits these valuesof a and g torsion angles in our structure is U105.Examination of the temporal changes of the U105torsion angles (Figure 7) reveals that conforma-tional changes of the c or a angles precede the flip.Similar changes in angular conformation of U105before the flip also occur in the PME calculations ofthe wild-type telomerase structure. Conformationalflexibility of U105 is affecting the dynamic proper-ties and positions of the neighboring nucleotidesconsequently translating the movement into thestructural fluctuation of the whole structure. There-fore, the U105 nucleotide is directly responsible for

Table 3. Average torsion angles of the wild-type hairpin structure computed over 20 ns

Torsionangles/residue Alpha P–O50 Beta O5 0–C50 Gamma C5 0–C40 Chi C1 0–N Delta C4 0–C3 0 Epsilon C3 0–O30 Zeta O30–P

StandardA-RNA

K68.0Kgauche

178.0trans

54.0Cgauche

K158.0anti

82.0Cgauche

K153.0trans

K71.0–gauche

U99 K74.38 174.89 65.54 K157.43 75.99 K155.51 K64.88U100 K74.77 174.83 63.95 K152.83 75.54 K162.01 K73.97U101 K74.63 176.03 63.12 K150.75 76.62 K159.84 K65.58U102 20.19 180.29 117.89 K153.39 77.14 K151.36 K68.80

K76.4(57%) 61.5(1%) 62.7(56%)147.9(43%) 181.1(99%) 188.9(44%)

U103 K75.96 173.60 61.77 K148.23 76.74 K159.18 K69.91C104 K38.18 178.32 82.13 K146.28 81.55 K131.03 K38.82

K76.5(83%) 60.2(83%) 80.5(97%) K158.1(64%) K54.3(86%)145.6(17%) 187.2(17%) 111.7(3%) K82.8(36%) 56.5(14%)

U105 39.30 123.64 186.51 K83.85 80.11 K168.47 K83.40K85.8(49%) 69.3(57%) K145.9(67%) 75.9(94%) K171.0(97%)160.7(51%) 194.8(43%) 40.9(33%) 148.0(6%) K79.0(3%)

C106 K82.41 172.96 56.77 K92.09 79.34 K150.64 K52.92K96.1(97%)

33.7(3%)G107 109.87 184.3 161.41 42.66 144.75 K81.71 80.35

K63.9(22%) 71.0(22%)159.8(78%) 187.4(78%)

C108 75.13 144.60 188.56 K156.28 81.77 K151.97 1.54K92.7(16%) 78.0(94%) K153.6(97%) K58.0(48%)188.(84%) 140.2(6%) K90.4(3%) 57.0(52%)

U109 K83.59 172.89 60.87 K101.62 126.10 K78.37 83.60K121.5(89%) 90.4(16%)

52.7(11%) 133.1(84%)G110 77.02 187.28 190.38 K168.50 80.12 K145.70 K67.21A111 K74.04 173.46 62.45 K159.76 76.85 K159.17 K61.01C112 39.68 180.46 127.06 K159.61 77.47 K147.04 K62.65

K74.9(50%) 64.0(50%)154.6(50%) 189.5(50%)

U113 K74.31 172.84 62.23 K153.06 75.60 K162.81 K74.92U114 K76.05 177.40 61.56 K150.64 75.57 K163.14 K66.22U115 58.08 179.55 145.62 K41.22 76.56 K150.18 K64.19

K75.6(38%) 53.7(3%) 62.8(36%) K138.3(41%)139.7(62%) 183.2(97%) 192.8(64%) 25.5(59%)

The first row represents the standard angles for an A-RNA helix.33 Bold font is used to highlight the unusual angles. If the angle haschanged from one conformation to another the relative average angles and the percentage of the trajectory in this conformation areprinted below the total average number.


the dramatic fluctuations of the hairpin structure,whereas the movements of the non-canonical bases,the least stable in the helix, are in response to themovement of the backbone. A stereo view of thelowest energy pentaloop structure is displayed inFigure 8(a). On the other hand, the presence of theconsecutive non-canonical base-pairs is crucial forthe existence of the flips, since the same dynamicalchanges in the torsion angles of the pentaloopregion were found in the test hairpin structure.However, without the mismatched base-pairs of thehelix the structure retains its stability, i.e. no flipsoccurred.

As an added test we performed GB MD simu-lations for 10 ns on the telomerase hairpin structurewhere the c angle of U105 base is restricted to ananti conformation. The RMSD shows considerableimprovement in overall stability with fluctuationsaround 1.5 A and no dramatic flips (Figure 9(a)).This observation again proves the importance of thetorsional changes in the U105 nucleotide on thehairpin dynamics.

The importance of the pentaloop residues

In view of the fact that the bases in the pentaloopare responsible for the fluctuations of the wholehairpin structure, one would suspect that mutationsof the bases in the pentaloop can lead to differentdynamical characteristics of the hairpin. Next, weexamine the effect of the DKC mutation in thepentaloop where G107 is replaced by A107 andC108 is replaced by G108 (Figure 1(c)). Examiningthe RSMD fluctuations (Figure 2(c)) we find thatthe structure is much more stable than the wild-type telomerase hairpin structure with a singlesignificant deviation from the average at 10.5 ns(point F) over a 20 ns time period. By mutating theresidues in the pentaloop sequence the stability ofthe hairpin structure significantly increases, indi-cating changes in the base-stacking interactions inthe pentaloop.

We compared the geometry of the pentaloop inthe DKC-mutated hairpin structure versus the wild-type hairpin structure (Table 4). The distance

Figure 7. Time-dependence of the torsional angles (a, b, g, c, d, 3, and z) of residue U105 of the wild-type hairpinstructure. Letters and vertical dotted lines denote the positions of the flips.


between U105 and U109 in the DKC-mutatedhairpin is now close enough to form a UU base-pair. In addition, the distance between U105 andG108 decreases enough to allow the possibility of a

GU wobble pair formation. Indeed, examinationof the trajectory reveals that in the DKC-mutatedhairpin structure U105 is now bonded to eitherG108 or U109. In the case of the wild-type hairpin

Figure 8. Stereo view of thelowest energy pentaloops of(a) the wild-type hairpin structureand (b) the DKC-mutated hairpinstructure. Blue are the U bases, redare the C bases, green are the Abases, and purple are the G bases.

Figure 9. RMSD versus time for(a) the wild-type hairpin structurewith the U105 c angle-constraint,(b) the A-hairpin structure, (c) theG-hairpin structure.


structure, the UU pair is not possible due to thelarge distance and the possible formation of a notvery stable UC pair. Therefore, in the DKC-mutatedhairpin structure, residue U105 is stabilized byhydrogen bonding and, thus, does not generate theflips. This agrees with experimental observationswhere the DKC mutant hairpin structure was foundto be more stable with the lowest energy structureindicating the possible formation of the GU wobblepair between U105 and G108.18 We performed MDsimulations on the structure used in this experi-mental study, which had the same pentaloopsequence as the DKC-mutated hairpin structurebut the helix consisted of canonical base-pairs. ThisDKC test hairpin structure displays the samedynamical interactions in the pentaloop with theU105 bonded to either G108 or U109. Moreover,examining the torsion angles (Table 5) we indeedfound that the U104/U105 region is a stable region.The central base in the pentaloop, A107, is nota “rigid nucleotide” but a very flexible one alongwith C106. These nucleotides, C106 and A107, aresticking out from the pentaloop in the DKCtelomerase (Figure 8(b)), whereas in the wild-typehairpin structure they are rotated into the pentaloopand C108 and U109 are out of the pentaloop.

We then checked for the existence of theconformational switches in the UU base-pairs andthe opening of the UC base-pair in the DKC-mutated hairpin when the pentaloop is stabilizedby the mutations and the structure displays morestability. By examining the direct hydrogen bondingdistances and occupancy (Table 1) we found that the

Table 4. Average distances between C1 0–C1 0 atoms in thepentaloop (A)

Base-pairsWild-type

hairpin, GBDKC-mutated

hairpin, GB

U105:U109 11.97 8.85C106:C/G108 5.49 8.96U105:C/G108 10.75 9.12U105:A/G107 9.27 9.99C106:U109 8.58 7.90A/G107:U109 7.75 8.69

conformational switches of the UU bases do exist inthe DKC-mutated hairpin structure. However, thereare fewer single-bond conformations than in thewild-type telomerase base-pairs. The UC base-pairis more stable and better aligned for hydrogenbonding. The U103:A111 base-pair has a fullhydrogen bond occupancy for 90% of the trajectoryand only 3% of the trajectory has the base-pairopened. The C104:G110 pair is very stable withalmost standard hydrogen bond occupancy (99%).

The question remains whether the mutation ofC108 or the mutation of G107 is more important tothe stability of the hairpin structure. The mutationof C108 to G108 in the pentaloop is responsible forthe existence of the hydrogen bond network in thepentaloop region. Whereas, the mutation of G107 toA107 relieves the rigid nucleotide constraint in thepentaloop, consequently allowing more freedomfor the base interactions. To investigate the relativeimportance of these mutations, we examined thedynamics of the two hairpin structures. The firsthairpin is the telomerase hairpin structure with asingle base mutation of G107 to A107 (A-hairpin);and the second structure has a single base mutationof C108 to G108 (G-hairpin). Comparison of theRMSDs for the A-hairpin (Figure 9(b)) and theG-hairpin (Figure 9(c)) structures depicts signifi-cantly increased stability in both cases as comparedto the wild-type hairpin structure, with G-hairpinstructure exhibiting slightly better stability. Thetrajectory of the A-hairpin structure shows variousperiodic hydrogen bond formations between U105and U109, U105 and C108, and C106 and U109. TheA107 base is a not a rigid nucleotide and thereforeallows more flexibility in the pentaloop to accom-modate for various hydrogen-bonding networks.The trajectory of the G-hairpin structure indicatesthe formation of the U105:G108 wobble base-pair.Yet G107 retains the behavior of a rigid nucleotide.

The dynamical flexibility of U105 is functionallycritical for the stability of the hairpin structure.Mutations of the residues within the pentaloop ofthe wild-type hairpin structure alter the base-pairing and base-stacking interactions in the

Table 5. Average torsion angles of the DKC-mutated hairpin structure computed over 20 ns

Torsionangles/residue Alpha P–O50 Beta O50–C50 Gamma C5 0–C40 Chi C1 0–N Delta C4 0–C30 Epsilon C3 0–O30 Zeta O3 0–P

U99 K74.12 177.81 64.17 K156.62 74.95 K157.89 K67.25U100 K39.61 176.55 83.55 K156.69 77.20 K152.06 K63.99

K75.4(84%) 63.4(84%)148.6(16%) 189.2(16%)

U101 K75.21 176.30 62.48 K153.02 75.55 K160.65 K72.55U102 K44.98 176.60 80.40 K149.53 75.95 K154.95 K70.77

K75.8(86%) 61.8(86%)146.0(14%) 188.1(14%)

U103 K77.34 171.64 62.20 K147.46 76.60 K159.20 K69.15C104 K76.90 171.87 64.32 K150.40 76.14 K156.21 K65.65U105 K74.18 172.77 65.56 K145.24 85.06 K166.23 K62.88

75.4(85%) K172.0(94%) K94.6(89%)140.4(15%) K83.2(6%) 195.5(11%)

C106 K11.04 176.14 58.79 K103.81 118.57 K108.63 K45.69K92.5(67%) 52.8(95%) 75.1(30%) K142.9(40%) K68.0(87%)154.7(33%) 180.3(5%) 137.2(70%) K86.5(60%) 96.6(13%)

A107 K40.19 180.53 64.27 K44.45 139.52 K72.77 87.36K65.0(85%) 44.3(85%) K106.9(58%) 91.0(3%)96.9(15%) 182.3(15%) 43.5(41%) 141.2(97%)

G108 64.52 187.84 186.73 K43.36 141.77 K80.23 107.52K161.3(3%) 54.3(3%)72.3(97%) 191.3(97%)

U109 17.73 184.32 149.11 K55.15 93.65 K134.26 K26.39K116.7(29%) 57.6(29%) K125.9(56%) 79.3(78%) K145.6(83%) K62.6(80%)

73.9(71%) 187.3(71%) 35.4(44%) 143.8(22%) K78.8(17%) 122.2(20%)G110 K71.29 169.31 63.63 K152.72 77.34 K165.60 K66.59

K82.2(98%) 61.6(98%)142.7(2%) 184.2(2%)

A111 104.82 187.52 160.38 K165.69 80.33 K149.88 K62.35K75.9(20%) 67.5(20%)149.3(80%) 183.1(80%)

C112 K73.39 176.67 64.13 K155.09 74.98 K157.39 K66.23U113 K32.55 176.26 87.53 K157.04 77.26 K151.56 K62.96

K75.5(81%) 64.0(81%)149.3(19%) 186.9(19%)

U114 K74.80 173.99 63.52 K144.18 75.23 K162.93 K74.69U115 K74.82 176.94 62.37 K151.26 75.46 K157.41 K69.62

Bold font is used to highlight the unusual angles. If the angle has changed from one conformation to another the relative average anglesand the percentage of the trajectory in this conformation are printed below the total average number.


pentaloop, which lead to a reduced flexibility ofU105 and hence a greater stability of the hairpinstructure. Therefore, the dynamical conformationalchanges of U105 may be an important feature thatis required for the binding of the hairpin structureto the P3 domain.

The proposed mechanism of pseudoknotformation

Based on our observations we propose a possiblemechanism for pseudoknot formation. As weobserved in our GB simulations, about every 4 nsthe wild-type hairpin structure undergoes remark-able structural rearrangements with a significantexpansion of the major groove and the opening ofthe non-canonical base-pairs. Moreover, at the flipsC112 is rotated outward from the helix creating anaccessible site for binding with the P3 domain. Inaddition, U113 and U114 are protruding into themajor groove, and in the pentaloop U109 and C108are also exposed on the same side as U114, U113,and C112. It is worth mentioning that this particularside of the pseudoknot structure will bind to the P3

domain (Figure 1(a)). Ultimately at the flip, thewhole non-standard region is open for hydrogenbonding with only the single C104:G110 base-pairholding together the pentaloop and the openregion. If one assumes that C112 binds with G178in the P3 domain of the telomerase RNA forming astable Watson–Crick GC base-pair, then this eventalone might be enough to break the hydrogen bondsof the C104:G110 base-pair, which is unstable at theflip. One may further assume that any otherprotruding base such as U113, U114, U109, orC108 forms a hydrogen bond with a base in the P3domain. At this point the energy will favorpseudoknot formation. Therefore, we propose thatthe periodic structural flips of the RNA hairpin cancreate nucleation points for pseudoknot formation.In 20 ns there are at least five distinct flips of thewild-type telomerase hairpin structure that mightact as starting points for pseudoknot formation. Inthe DKC-mutated hairpin structure, during 20 nsonly one structural opening of the non-standardregion was detected. Therefore, the DKC mutationsin the hairpin structure reduce the probability ofthe possible formation of the pseudoknot. This


assumption can be supported by experimentalobservations where the wild-type pseudoknotdominates the hairpin structure approximately95% to 5%, respectively, while the DKC mutationleads to a shift in equilibrium to about 50/50.17

However, it is unknown whether the 50/50 equi-librium in the DKC-mutated telomerase RNA hair-pin occurs due to the instability of the pseudoknotor stability of the hairpin structure, although ourresults suggest the latter.

Conclusions

We have performed and analyzed variousmolecular dynamics simulations of the telomeraseRNA hairpin structure with and without differentmutations. We found that the wild-type telomeraseRNA hairpin is a very flexible structure withremarkable periodic structural rearrangements orflips. When the flips occur, the region with non-canonical base-pairs loses the hydrogen-bondingnetwork, creating a possible site for binding withother domains. Based on the observed temporaldynamical changes in the human telomerase hair-pin structure, we propose that these periodic flipscan possibly produce a nucleation point for bindingwith the P3 domain and eventually lead to theformation of the pseudoknot. The similarity of theobserved results between explicit and implicitsolvent models reveals that explicit water moleculesand ions do not significantly impact the dynamicalcharacteristics of this hairpin structure.

Moreover, we have found that the rearrangementof nucleotide U105, which is located in thepentaloop region, induces the flips. Mutations inthe pentaloop, as in the case of the DKC mutations,stabilize the structure by preventing the flexibilityof U105 consequently creating a hydrogen bondnetwork in the pentaloop. However, the consecu-tive non-canonical base-pairs are very importantfor the observed structural deviations since theirinstability permits the widening of the major grooveof the hairpin structure and the opening of the base-pairs, which can possibly act as binding site for theP3 domain of telomerase RNA.

Modeling Method and Structures

In this study we present the results from eightseparate molecular dynamics simulations on sixdifferent RNA molecules as described below.

(1)
The most studied structure in this paper is theRNA hairpin structure of the wild-type telo-merase (Figure 1(b) and (e)). The startingcoordinates were taken from the experimentalNMR structure (PDB databank entry: 1NA217)and will be referred to as the wild-type hairpinin the text.
(2)
The DKC mutation in the telomerase hairpinstructure (Figure 1(c)). The 3D structure is
produced by replacing G107 and C108 withA107 and G108 in the wild-type hairpinstructure and will be referred to as the DKC-mutated hairpin.

(3)
The structure of the test-case hairpin (Figure1(d)). Here the non-canonical U-U and U-Cbase-pairs are replaced by standard Watson–Crick base-pairs. The stem with standardWatson–Crick bases is created with Insight IIwand capped with the UCUCGCUGA sequence(the pentaloop and two base-pairs) from thewild-type hairpin structure. This structure willbe called the test hairpin.
(4)
The 15-base DKC-mutated test hairpin structurewas taken from the experimental NMR struc-ture (PDB databank entry: 1Q7518) where thepentaloop sequence is the same as in DKCtelomerase hairpin structure; however, there areno non-canonical base-pairs in the helix. Thisstructure will be referred to as the DKC testhairpin.
(5)
The hairpin structure with one base mutationwhere G107 is replaced by A107. This structurereferred to as the A-hairpin.
(6)
The hairpin structure with one base mutationwhere C108 is replaced by G108. This structurewill be referred to as the G-hairpin.
An accurate description of the force field can playa crucial role on the dynamical behavior of nucleicacids. All simulations were performed using ff99Cornell force field for RNA,37 which has proven tobe a reliable and refined force field for nucleic acids,and the molecular dynamics software Amber 7.0.38

Nucleic acids are highly charged molecules; there-fore, electrostatic interactions that define the sol-vation energies need to be carefully calculated.There are two well-known approaches for calcu-lating solvation energies: explicit solvation modelsand implicit solvation models. The explicit solventmethod produces the most accurate modelingof solvation effects and can provide crucial infor-mation on direct interactions of water and ions withnucleic acids.29 The disadvantage of this method isthat it is slow and computationally expensive due tothe addition of thousands of water molecules thatincreases both the system size and the simulationtime. Implicit solvation methods39 are based on theapproximation of electrostatic solvation free ener-gies assuming that the solvent is a uniform high-dielectric constant medium that responds to thepartial charges of a low-dielectric constant solute.Implicit solvation methods significantly reducecomputational time and storage requirements,therefore, permitting longer computational timesand larger molecules to be studied. These methodshave proven to be reliable and able to providecrucial information for various biomolecules.40,41

The GB theory42–44 is one of the most successfulapproximations of the Poisson equation for con-tinuum electrostatic solvation energy and describesthe electrostatic energy of two or more atomiccharges in a cavity of arbitrary shape. It involves


accurate evaluation of Born radii, which charac-terize the average spherical distances of each atomto the solvent boundary. Consequently, the GBenergy expression involving summation of self andpairwise interactions of atomic charges is ananalytical function of atomic positions and is verygood at reproducing the Poisson energy.

The simulations using the GB implicit solventapproach as implemented in the Sander module ofAMBER were performed for all structures. Eachstarting structure was subjected to minimization(5000 steps), followed by slow 20 kcal/mol con-strained heating to 300 K, and several consecutiveMD equilibrations with declining constraints from2 kcal/mol to 0.1 kcal/mol over a total 150 ps timeperiod. The temperature was maintained at 300 Kusing a Berendsen thermostat.45 The monovalentsalt concentration was set to 0.5 mol/l. The pro-duction simulations were performed for 10 ns or20 ns using 1 fs time step. An additional GBcalculation of the wild-type hairpin structure wasperformed with a c angle constraint of the U105nucleotide for a 10 ns simulation time.

In addition, the wild-type hairpin structure wascalculated using explicit water and ions. Theelectrostatic interactions were calculated by PMEsummation46 and the non-bonded interactions weretruncated at 9 A. For a 30-base RNA hairpinmolecule 29 KC counterions and 25 KC/ClK pairswere used with 10,443 TIP3P water molecules tosolvate the RNA molecule to a 0.1 mol/l relative saltconcentration. The final box dimension was 63 A!63 A!82 A and the total system consisted of 32,399atoms. The system was minimized constrainingthe solute then solvent, then heated to 300 Kconstraining the RNA then the solute, and finallyequilibrated by slowly releasing the constraints.SHAKE was applied to all hydrogen bonds in thesystem. Pressure was maintained at 1.0 Pa using theBerendsen algorithm, and a periodic boundarycondition was imposed. A production simulationwas performed for 20 ns with a 2 fs timestep.

All simulations were carried out on an SGI-Altixand SGI-Origin computers using four or eightprocessors. The analysis for all simulations wasperformed using the Carnal and Ptraj modules onthe production simulations excluding the initialequilibration stage.

Acknowledgements

This work was supported by the Laboratory ofExperimental and Computational Biology. Thecomputational support was provided by theNational Cancer Institute’s Advanced BiomedicalComputing Center.

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Edited by M. Levitt

(Received 16 December 2004; received in revised form 31 January 2005; accepted 4 February 2005)

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