The Sarcin / Ricin Loop, a Modular RNA€¦ · The Sarcin/Ricin Loop, a Modular RNA A. A. Szewczak and P. B. Moore* Departments of Chemistry The conformation of a 29 base oligonucleotide

JMB—MS 326 Cust. Ref. No. PEW 137/94 [SGML]

J. Mol. Biol. (1995) 247, 81–98

The Sarcin/Ricin Loop, a Modular RNA

A. A. Szewczak and P. B. Moore*

The conformation of a 29 base oligonucleotide called E73 has beenDepartments of Chemistrydetermined in solution by NMR. E73 includes a 23 nucleotide sequence thatand Molecular Biophysics and

Biochemistry, Yale University is identical with that of a the a-sarcin and ricin-sensitive loop (SRL) fromrat 28 S rRNA, and like the SRL in intact ribosomes, E73 is a substrate forNew Haven, CT 06520

U.S.A. both toxins. The SRL includes a long, conserved sequence found in theRNA of all large ribosomal subunits, which plays a critical role in thefactor-dependent steps of protein synthesis. The spectroscopic observationsand analysis that led to the determination of the conformation of E73 arepresented. The SRL in E73 has a highly structured conformation, which isstabilized by several non-Watson-Crick base-pairs, and many properties ofthe SRL in the ribosome can be understood assuming that the conformationof E73 and that of the SRL in the ribosome are the same. The role of the SRLin protein synthesis is discussed in light of the conformation of E73, as is themodular relationship that exists between the conformation of the SRL andother smaller RNAs.

Keywords: ribosomal RNA; NMR; ricin; alpha sarcin; structure*Corresponding author

Introduction

The longest conserved ribosomal RNA sequence,5'AGUACGAGAGGA3', is found in the principalRNA of large ribosomal subunits (see Gutell et al.,1992): A2654 to A2665 in Escherichia coli 23 S rRNA(Noller et al., 1981), and A4318 to A4329 in rat 28 SrRNA (Endo & Wool, 1982). It is embedded in asomewhat longer sequence named the sarcin/ricinloop (SRL) because it is the site of attack of twoprotein toxins, ricin and a-sarcin, that kill cells byinactivating ribosomes.

Ricin catalyzes the depurination of the third Afrom the 5' end of this sequence (Endo et al., 1987),while a-sarcin catalyzes the hydrolysis of thephosphodiester bond that links its eighth and ninthresidues (Endo & Wool, 1982). Once damaged thisway, ribosomes do not bind elongation factorsproperly (Hausner et al., 1987), and that failureresults in the cessation of protein synthesis.

Consistent with these findings, the SRL is protectedfrom chemical modification when EF-G and EF-Tubind to the ribosome (Moazed et al., 1988).

The study reported below was motivated by thediscovery that a-sarcin and ricin cleave oligoribonu-cleotides having the SRL sequence in vitro (Glucket al., 1992). The loop sequence, in either itsprokaryotic, 15 residue form or its eukaryotic, 17base form, when closed by a short helix of arbitrarysequence is a substrate for both enzymes. The RNAoligonucleotide whose conformation is discussedhere, E73 (Figure 1), has a eukaryotic 17 base loop.

Earlier studies demonstrated that the SRL ishighly structured (Szewczak et al., 1991), and aconformational model was published based on apreliminary analysis of spectroscopic data(Szewczak et al., 1993b). Neither the spectroscopicdata nor the assignments on which that analysisdepended were presented, however. Both aredescribed below, and a final, refined model for themolecule is shown and discussed.

Results

Imino proton assignments

Figure 2(a) shows the imino proton spectrum ofE73, taken in H2O at 283 K. At both 283 K and 303 K,it is easy to detect NOEs that link resonances D, B,A, F and C sequentially, and show that they representthe imino protons in the stem of E73 (G2 throughG24, respectively: Szewczak et al., 1991). At lowtemperatures, an additional run of NOEs can be

Current address: A. A. Szewczak, Department ofChemistry and Biochemistry, University of Colorado,Boulder, CO 80309, U.S.A.

Abbreviations used: SRL, sarcin/ricin loop; EF,elongation factor; p.p.m., parts per million; NOE,nuclear Overhauser enhancement; NOESY, NOEspectroscopy; COSY, correlated spectroscopy; DQF,double quantum filtered; TOCSY, total correlationspectroscopy; TPPI, time-proportional phaseincrementation; HMQC, heteronuclear multiplequantum correlation; GARP, globally optimizedalternating phase rectangular pulse; FID, free inductiondecay; r.m.s.d., root-mean-square difference.

0022–2836/95/110081–18 $08.00/0 7 1995 Academic Press Limited

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The Sarcin/Ricin Loop82

Figure 1. RNA sequences. The sequences of the 2 RNAsexamined in this study, E73 and E73-S, are shown here.Residues A9 to A20 correspond to the conserved sequenceof the sarcin/ricin loop. The adenine depurinated by ricinis designated an asterisk (*).

The aromatic/anomeric walk

For an RNA of its size, E73 has a remarkablywell-resolved 1H spectrum (Figure 2(b)). Itspyrimidine H(5) and H(6) resonances were identifiedon the basis of their H(5)/H(6) correlations. There is asingle crosspeak for each of the 12 pyrimidineresidues in COSY spectra of E73, which indicates thata single conformation of E73 dominates under theseconditions (data not shown). The three U residueswere distinguished from the eight C residues byrepeating the COSY experiment using an E73 samplelabelled with deuterium at C5.

Sequence-specific assignments were made forthese and all the other anomeric and aromaticprotons of E73, at both 283 K and 303 K, on the basisof the NOESY spectra given by three samples:protonated E73, protonated E73-S, and E73 deuter-ated at both the 8 position of G residues and the 5position of C residues. In this way stem resonancescould be distinguished from loop resonances, and A,G, C and U residues positively identified.

Partial deuteration affected the relaxation proper-ties of E73 resonances in the manner proteinspectroscopists have described in the past (LeMaster& Richards, 1988). The dG8,dC5 sample gave sharperNOEs than its unlabelled counterpart, and relativeNOE intensities changed in response to thealterations in relaxation pathways that resulted fromdeuteration. The dG8,dC5 sample’s NOESY spectrathus proved more illuminating than we hadanticipated.

Figure 3(a) shows the anomeric/aromatic part ofE73’s NOESY spectrum with the anomeric/aromaticwalks from G1 to A9, and from C22 to C29superimposed on it. At A9, the continuity stops;there is no A9 to G10 NOE in the anomeric/aromaticregion. Providentially, two of the three U residues in

detected that connect H1, the downfield componentof H, with E2, the upfield component of E, on to G,then E1, the downfield component of E, and finallyback to H2, the upfield component of H. This groupof resonances, which must come from the loop, wereassigned on the basis of a natural abundance 1H-15Ncorrelation experiment (data not shown). Thisexperiment demonstrated that all the resonances inthe loop set are GN1 resonances except E1, which isa UN3 resonance (Szewczak et al., 1993a). (It alsoconfirmed that resonance A is a UN3 resonance, asexpected.) The only U in the loop that could have Gresidues with unassigned imino proton resonance onboth sides is U11. H1, E2, G, E1 and H2, therefore,must correspond to G14, G18, G19, U11 and G10,respectively (see Table 1).

The low-temperature imino proton spectrum ofE73 includes a broad resonance at about 10.6 p.p.m.,resonance I. It could belong to either U7 or G16, butit gives no NOE, and its 1H-15N crosspeak, whichwould have identified it, was not detected.

(a)

Figure 2. E73 proton spectra. (a)Imino proton spectrum. This spec-(b) trum was collected in H2O at 283 Kusing the twin pulse method forwater suppression (Kime & Moore,1983a). Resonances are identified byletters for ease of identification(Szewczak et al., 1991). (b) Non-ex-changeable proton spectrum. Thisspectrum was collected at 303 Kin 2H2O, as described in Materialsand Methods. The upfield-mostresonance belongs to EDTA, and thatimmediately downfield of it is that ofdioxane, the chemical shift standard.The 1H2HO resonance at about4.7 p.p.m. is off-scale.

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The Sarcin/Ricin Loop 83

(a)

Figure 3. The anomeric-aromaticregion of the NOESY spectrum ofE73. A NOESY spectrum is shown,which was taken in 2H2O at 303 Kwith a 600 ms mixing time. (a) TheE73 anomeric-aromatic walk for G1to A9 and C22 to C29 is superim-posed on the spectrum. Continuouslines trace the anomeric-aromaticwalk for E73 residues G1 to A9, anddotted lines show the connectivitiesfor C22 to C29. The cross strand NOEinvolving the H(2) of A26 is markedwith a broken line. (b) The E73anomeric-aromatic walk for U11 toC22 is traced out with a continuousline. The H(6) chemical shift for U11 ismarked with a broken line, and someimportant, non-helical NOEs are alsoindicated.(b)

E73 lie between G1 and A9, so that U11 could beassigned by elimination, and the sequential walkresumed. The only G10-related sequential NOEimmediately apparent is the G10H(1') to U11H(6) NOEat (5.96 p.p.m./7.96 p.p.m.). That NOE is obviouslya doublet in the H(1') dimension, which indicates thatthe ribose of G10 is in the S conformation, which ischaracterized by large (H(1'), H(2')) couplings. Inaddition, there is a strong G10H(1')-U11H(5) NOE(Figure 4(a)). Inexplicably, we were unable to detecta G10 (H(8)/H(1')) NOE, and we are still unsure of thechemical shift of G10H(8). All of the GH(8) resonancesidentified by comparing the spectra of dG8, dC5 E73and protonated E73 are securely assigned to otherresidues.

Figure 3(b) shows the anomeric/aromatic walkstarting at U11, and ending at C22. It breaks into threesegments: U11 to C13, G14 to A17, and G18 to C22.The break between C13 and G14 is not a real one.C13’s H(1') does give a normal, sequential NOE to G14’sH(8), but it is hard to identify because the chemicalshift of C13H(1') is 4.10 p.p.m., a full p.p.m. upfield ofnormal. The break between A17 and G18, on theother hand, is real. The H(1') proton of A17 does notgive an NOE to the H(8) of G18. What is seen instead(at long mixing times) is the reverse, an A17H(8)/G18H(1') NOE, and its recognition was complicated bythe unusual chemical shift of G18H(1'), 3.98 p.p.m.

All of the intraresidue H(1')/(H(6) or H(8)) NOEs inE73 NOESY spectra are much weaker than

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(a)

(b)

Figure 4. The anomeric and aromatic regions of the E73NOESY spectrum. This figure shows the near-diagonalregion of the same NOESY spectrum whose anomeric-aro-matic region is shown in Figure 3. (a) The anomeric neardiagonal. Several NOEs involving anomeric and H(5)

protons are labelled. (b) The aromatic near diagonal. Allimportant NOE crosspeaks are labelled.

Four of the six remaining AH(2) protons give NOEsto the 1' protons of their 3' neighboring residue:A12, A16, A20 and A21. The A20H(2)/A21H(1') NOE isalmost as intense as a pyrimidine H(5)/H(6) NOE,which indicates that those protons are unusuallyclose together. A17H(2) and A9H(2) were revealed byweak NOEs to their own H(1') protons at long mixingtimes. NOEs of this kind must be the productsof multiple transfers of magnetization, e.g.nH(2) : (n + 1)H(1') : (n + 1)(H(6) or H(8)) : nH(1'),but in our experience, they are often observed.

Non-anomeric sugar protons

Twenty-four of the 29 H(2') resonances in E73 wereidentified on the basis of strong NOEs to 1' protonsseen in short mixing time NOESY spectra. The H(2')

protons of C13 and G18 could not be picked up thisway because their H(1')/H(2') NOESY peaks lie in thedensely populated ribose-ribose region. A9, G10 andU11 also give H(1')/H(2') NOEs in the anomeric/riboseregion, but they are surprisingly weak. The H(2')

assignments for A9 and G10 were confirmed byDQF-COSY spectra. The 1' protons of both residuesgive conspicuous COSY peaks in the ribose region(Figure 5(a)), which indicates that both residues arein the S conformation, an inference already reachedfor G10 on the basis of NOESY data. The H(1')/H(2')

couplings in residues G1, A15, G16, A17 and C29 arealso large enough to result in detectable COSY peaks,which indicates that their ribose conformations are amixture of N and S. The ribose rings of the remaining22 residues are in the N conformation.

Despite the prevalence of the N ribose residues inE73, the anomeric/ribose region of E73 TOCSYspectra was extremely informative (Figure 5(b)). H(1')

to H(2') to H(3') TOCSY connectivities were evident forall residues except C13 and G18, and theseconnectivities could be followed out to H(4') for 22 ofthe 29 residues. Further sugar assignments resultedfrom 1H-31P hetero-TOCSY experiments (see below).The ribose spin systems of C13 and G18 wereassigned in that way, as were some of the H(4')

assignments and most of the (H(5'), H(5")) assignmentsobtained.

The COSY spectrum of E73 includes a crosspeakat (5.75 p.p.m., 4.78 p.p.m.) that could not beassigned. Although its multiplet structure indicatesa coupling constant larger than that of the nearby A15crosspeak, its intensity is weaker. We believe that thiscrosspeak is indicative of the existence of a smallpopulation of the molecules in the sample that are inan alternative conformation. There is no NOE peakat that position.

1H-13C correlations

Figure 6 is a natural abundance 1H-13C HMQCspectrum of E73. Its aromatic correlations are aliasedbecause the carbon sweep-width was reduced tomake data collection efficient. The most important

pyrimidine H(5)/H(6) NOEs. Since the intensities ofintraresidue H(1')/(H(6) or H(8)) NOEs approximatethose of H(5)/H(6) NOEs only when nucleotides are inthe syn conformation, all the residues in E73 must bein the anti conformation.

Assignment of AH (2) resonances

AH(2) resonances in E73 were identified as agroup on the basis of both T1 measurements,their longitudinal relaxation times are abouttwice as long as those of other aromatic resonances(seven to eight versus three to four seconds),and by natural abundance 1H-13C spectroscopy.One of these slow relaxing resonances, that at7.43 p.p.m., had already been identified as theA26H(2) by imino proton spectroscopy; it is thearomatic correlate of U4H(1). It also gives NOEs to the1' protons of C27 and G5, as expected for an AH(2) ina helical stem.

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(a)

Figure 5. DQF-COSY and cleanTOCSY spectra of E73. DQF-COSYand Clean TOCSY spectra weretaken at 30°C using the d5C,d8G E73sample, as described in Materialsand Methods. (a) The anomeric-ri-bose region of E73’s DQF-filteredCOSY spectrum. The H(1')-H(2')

crosspeaks observed are labelled byresidue number, and some ad-ditional scalar correlations are indi-cated. (b) The anomeric-ribose regionof E73’s clean TOCSY spectrum.Many more H(1') correlations are seenin this spectrum than in its DQF-

(b) COSY counterpart.

correlations in this spectrum are those assignedto the 1' protons of G10, C13 and G18. Thepeanut-shaped peak labelled ‘‘C13, G18’’ has a 13Cchemical shift in the H(1') range, and its 1H chemicalshift(s) are those already assigned to the H(1') protonsof C13 and G18. The assignments of the 1' protons ofC13 and G18 to upfield resonances are thusconfirmed.

The peak belonging to G10H(1') is more difficultto assign. Its 1H chemical shift was assigned earlierto G10H(1'), 5.96 p.p.m., but its 13C chemical shift,81 p.p.m., is more appropriate for an H(4') resonancethan an H(1') resonance. COSY and TOCSY datashow that the 5.96 p.p.m. resonance is part of aspin system that includes resonances at 4.89and 4.95 p.p.m., in the order 5.96, 4.89, 4.95. Inaddition, there are only two 1H/13C peaks in Figure 7with 1H chemical shifts of 4.95 p.p.m. On thebasis of 13C chemical shifts, one of them is likelyto be a 4' proton and the other a 2' or 3' proton.Furthermore, 1H-31P COSY data indicate thatboth proton resonances are coupled to phosphorus,which the 5.96 p.p.m. resonance is not. Thus the4.95 p.p.m. resonance in the 5.96 p.p.m. spin systemclearly is not a 1' resonance, nor a 2' resonance. If it

is assigned to G10H(4'), then the 5.96 p.p.m. resonanceis either a 2', a 5' or a 5" resonance. These alternativeswould imply that the 13C chemical shift of the5.96 p.p.m. resonance is even more anomalous thanit is already, assigned as a 1' resonance. Theassignment we prefer, namely that the 4.95 p.p.m.resonance in the 5.96 p.p.m. spin system representsG10H(3'), appears to be the only viable alternative.The resonance at 5.96 p.p.m. must be G10H(1'),despite its unusual 13C chemical shift. It should benoted that C(1') carbon chemical shift depends onsugar pucker, and C(2')-endo conformation is associ-ated with upfield 13C chemical shifts (Varani &Tinoco, 1991b). Why this particular C(1') has achemical shift so far out of the expected rangeremains to be explained.

Phosphorus assignments

Experiments of the 1H-31P hetero-TOCSY classwere used to assign E73’s 31P spectrum, which isexceptionally well dispersed (see Szewczak et al.,1991). The assignments of the outlying phosphorusresonances are indicated in Figure 7. They fallinto two connected groups, one centered around

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Figure 6. E73’s 1H-13C HMQCspectrum. A natural abundance1H-13C HMQC was taken of E73, inwhich the spectrum is aliased so thataromatic correlations appear unnatu-rally far upfield in the 13C dimension.Aromatic crosspeaks are partialmultiplets because of incompletedecoupling of downfield protons (seeMaterials and Methods). The 2 H(1')

resonances that are furthest upfieldin 1H chemical shift are indicated.Attention is called to the crosspeak at81.3 p.p.m. (13C), 5.96 p.p.m. (1H). Ittoo is an H(1') resonance (see the text).

the residues attacked by ricin and a-sarcin,G14pA15pG16pA17pG18, and the other in theneighborhood of A9, the place where the first breakin sequential connectivities occurs, U7pC8pA9pG10.The conformation of the backbone must be unusualin both regions.

Both two and three-dimensional hetero-TOCSY-NOESY experiments were run to confirm theassignments made on the basis of hetero-TOCSYdata, and to extend assignments in ribose spinsystems. Experiments of this class generate 31Pcorrelations with anomeric and aromatic protons,which is particularly useful since anomeric andaromatic assignments tend to be secure. Figure 8shows a plane of constant 31P chemical shift from athree-dimensional hetero-TOCSY-NOESY spec-trum. Correlations involving U11’s H(1'), H(2'), H(3') andH(4') are clearly evident, as is a NOESY crosspeakinvolving A12H(8).

Tables 1 and 2 summarize the proton assignmentsthat have emerged for E73 at 283 K and 303 K, whileTable 3 provides the phosphorus, carbon andnitrogen assignments obtained.

The hydrogen-bonding pattern in the loop

The hydrogen-bonding pattern in the loop of E73can be deduced from the large number of NOEsbetween imino protons and other protons observedin its water NOESY spectra. The easiest ones tounderstand are those involving the imino proton of

G18, which gives NOEs to the exocyclic aminoprotons of C13, and to C13H5. They can be explainedby proposing that G18 and C13 form a Watson-Crickbase-pair (see Figure 9). All its other NOEs relate toG19. Once G18 and C13 are paired, G14 aligns withA17. The only G14 imino to A17 NOE involvesA17H8, which suggests an AG pair of the typeshown in Figure 9, which is the only anti-antipairing possible consistent with that NOE. Theimino proton of G19 gives a weak NOE to theH8 of A12, the base that aligns with it, giventhe G18-C13 pair. This suggests another AGbase-pair of the G14-A17 type. There is a strong NOEbetween U11H3 and A20H8, the base U11 alignswith, and the only anti-anti base-pair compatiblewith this NOE is a reversed-Hoogsteen AU pair(Figure 9).

The most unusual imino-other NOEs in the setare those between G10H1 and the H(2') and H(3')

protons of G19. They indicate that G10 reachesacross the major groove of the loop all the wayto the backbone of the opposite strand. If G10is a bulged residue, then the base-pair belowU11-A20, if it exists at all, should involve A9 and A21.C8 and C22 will also lie opposite each other, as willU7 and C23. The evidence available about theorganization of this part of the molecule is slender.There is no U7 imino proton NOE, and no cross-strand NOE involving non-exchangeable protonswhatever. The only hint we have comes from an NOEbetween A21H8 and a pair of amino protons that are

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Figure 7. E73 1H-31P heteroTOCSYspectrum. A 1H-31P heteroTOCSYspectrum was collected at 303 K witha mixing time of 24 ms, as describedin Materials and Methods. The entirespectrum is shown, and the assign-ments of the outlying 31P resonancesare given.

likely to belong to A9. This is consistent with thehypothesis that A9 and A21 form a symmetric A-Apair (Figure 9), but is less than proof; the A9H(8) toA21NH NOEs one expects have not been identified.

Non-sequential loop NOEs

Non-sequential NOEs between non-exchangeableprotons are rare in RNAs, but very revealing whenthey occur. Fortunately there are several in theloop, and with the exception of the strongA12H(2)/A20H(2) NOE (Figure 4(b)), they all speak tothe conformation of the molecule in the neighbor-hood of G10. For example, one of the strongest piecesof evidence that G10 is a bulged base is an NOE

between A9H(8) and U11H(6) seen at long mixingtimes (data not shown). It is the only non-sequentialaromatic/aromatic NOE detected, besides A12H(2)-A20H(2), and it proves that the A9-A21 pair stacks onthe U11-A20 pair.

NOEs are also detected that correspond toG10H(1')/U11H(5), A9H(1')/G10H(1') and A9H(1')/U11H(5) (Figure 4(a)). These correlations explain abaffling feature of the aromatic-anomeric spectrum.Even though A9 and U11 are non-sequential,the A9H(8)/A9H(1') crosspeak and the U11H(6)/U11H(1') crosspeak, which has a strange, multi-plet-like appearance, line up with each other atall temperatures, and in some spectra, look evenmore alike than they do in Figure 3. The explanation

Figure 8. The U11 Slice of a 1H-31P3-dimensional heteroTOCSY-NOESY spectrum. A 3-dimensionalheteroTOCSY-NOESY spectrum ofE73 was obtained at 303 K (seeMaterials and Methods). The planeof constant 31P chemical shift isshown that includes U11pA12. Someof the more obvious correlations areidentified. Most of the correlationsunrelated to U11 come from C28.

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Table 1E73 1H assignments (10°C)Residue H(6)/H(8) H(2)/H(5) H(1') H(2') H(3') H(4') H(5') and H(5") Imino NH(1) and NH(2)

G1 8.17 na 5.80 4.92 4.69 4.55 — — — — —G2 7.55 na 5.93 4.72 4.58 4.01 — — 12.89 (8.40) (6.03)G3 7.21 na 5.78 4.48 4.44 4.37 — — 13.21 — (6.08)U4 7.73 5.07 5.55 4.63 4.53 4.44 — — 13.68 na naG5 7.65 na 5.76 4.51 4.44 — — — 12.66 (8.08) (5.94)C6 7.20 5.01 5.26 4.51 3.97 4.31 — — na 8.24 6.98U7 7.46 5.19 5.36 4.13 3.98 4.17 — — — na naC8 7.37 4.69 5.36 4.14 (3.99) — — — na — —A9 8.61 7.70 5.68 3.99 4.65 — — — na 8.72 6.72G10 — na 5.99 4.90 — 4.47 — — 10.18 — —U11 7.96 5.90 5.66 (4.55) 4.80 (4.15) — — 12.79 na naA12 7.51 6.94 5.74 4.69 4.50 4.33 4.15 4.01 na — —C13 7.41 5.65 4.10 4.14 4.32 — — — na 8.30 7.31G14 7.21 na 5.67 4.22 4.54 4.78 — — 10.20 — —A15 8.25 7.85 5.66 4.77 4.33 4.16 4.23 3.86 na — —G16 7.44 na 4.95 4.17 4.40 4.22 3.86 3.81 — — —A17 8.08 (7.66) 6.04 4.37 4.41 4.95 (4.05) (3.77) na — —G18 7.60 na 4.16 3.85 4.05 4.48 — — 12.76 — —G19 7.47 na 5.16 4.27 4.71 4.40 (4.56) (4.08) 11.50 8.14 —A20 7.54 7.89 5.98 5.13 (4.22) 4.85 — — na 6.45 6.82A21 7.84 7.83 5.58 4.32 4.51 4.69 4.16 — na — —C22 7.36 5.21 5.78 4.37 — — — — na — —C23 7.38 5.56 5.62 4.45 4.27 4.40 — — na — —G24 7.37 na 5.60 4.56 4.41 — — — 12.98 (8.52) (6.42)C25 7.74 5.22 5.48 4.48 4.59 4.42 — — na 8.60 7.02A26 8.08 7.42 5.93 4.54 4.71 4.49 4.36 4.13 na (8.11) (6.36)C27 7.56 5.22 5.38 4.22 4.36 — — — na 8.45 7.04C28 7.76 5.43 5.48 4.24 4.47 — — — na 8.55 6.90C29 7.66 5.47 5.72 3.99 4.16 4.78 — — na 8.32 7.02

na, Not applicable; no, not observed; ( ) denotes tentative assignment; 5' and 5"assignments are not stereospecific. Chemical shifts aregiven in p.p.m. relative to a dioxane chemical shift standard (3.741 p.p.m.), and are reproducible to about 20.02 p.p.m.

Table 2E73 1H assignments (30°C)

J1'2'

Residue H(6)/H(8) H(2)/H(5) H(1') H(2') H(3') H(4') H(5') and H(5") (Hz) H(1)/H(3) NH(1) and NH(2)

G1 8.17 na 5.83 4.93 4.69 4.54 4.24 4.42 3.7 no — —G2 7.54 na 5.93 4.75 4.53 4.04 (4.24) — 0-2 12.84 — —G3 7.21 na 5.78 4.46 4.43 4.32 4.09 4.27 0-2 13.19 — —U4 7.69 5.07 5.56 4.62 4.51 4.42 4.07 4.23 0-2 13.60 na naG5 7.63 na 5.76 4.52 4.40 4.45 (4.07) — 0-2 12.62 — —C6 7.21 5.01 5.28 4.50 3.97 4.31 4.07 4.38 0-2 na 8.20 6.83U7 7.44 5.19 5.36 4.08 3.96 4.17 3.87 4.25 0-2 no na naC8 7.34 4.71 5.37 4.14 4.32 4.12 4.01 4.43 0-2 na — —A9 8.54 7.70 5.66 3.99 4.63 3.89 4.12 4.36 8.5 na — —G10 no na 5.96 4.89 4.95 4.43 4.10 4.21 9.9 10.20 — —U11 7.96 5.89 5.63 4.53 4.78 (4.14) — — 0-2 12.75 na naA12 7.54 6.94 5.73 4.67 4.46 (4.53) 4.08 — 0-2 na — —C13 7.42 5.65 4.10 4.16 4.28 4.00 4.14 4.24 0-2 na 8.20 7.20G14 7.30 na 5.62 4.25 4.52 — 4.00 — 0-2 10.20 — —A15 8.20 7.91 5.68 4.67 4.34 4.17 3.84 4.21 4.6 na — —G16 7.51 na 5.06 4.21 4.43 4.19 3.82 3.97 4.4 no — —A17 8.10 8.18 6.05 4.44 4.95 4.41 4.00 3.90 4.3 na — —G18 7.58 na 3.98 4.24 3.90 4.51 4.11 4.25 0-2 12.76 — —G19 7.51 na 5.17 4.28 4.71 4.39 — — 0-2 11.53 — —A20 7.55 7.91 5.98 5.12 4.22 4.85 4.53 — 0-2 na 6.72 (5.95)A21 7.82 7.84 5.57 4.31 4.66 4.48 4.13 4.30 0-2 na — —C22 7.37 5.15 5.78 4.36 4.34 (4.35) (4.08) 4.58 0-2 na — —C23 7.38 5.55 5.59 4.45 4.25 4.39 (4.09) — 0-2 na — —G24 7.39 na 5.59 4.57 4.41 (4.26) — — 0-2 12.96 — —C25 7.73 5.24 5.49 4.48 4.56 4.42 4.07 4.27 0-2 na 8.54 6.92A26 8.06 7.43 5.93 4.54 4.66 4.48 4.12 4.29 0-2 na — —C27 7.54 5.22 5.39 4.22 4.35 (4.32) 4.03 — 0-2 na 8.34 6.93C28 7.76 5.43 5.49 4.24 4.46 4.36 4.01 — 0-2 na 8.51 6.81C29 7.66 5.47 5.75 3.99 4.17 (4.76) 4.05 4.16 3.6 na — —

na, Not applicable; no, not observed; ( ) denotes tentative assignment; 5' and 5" assignments are not stereospecific.Chemical shifts are reported in p.p.m. relative to a dioxane chemical shift standard, and are accurate to about 20.02 p.p.m.

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Table 3E73 13C, 15N and 31P assignmentsRes. C(6)/C(8) C(2)/C(5) C(1') C(2') C(3') C(4') N(1)/N(3)

31P

G1 136.4 na 89.0 72.5 — 80.8 — naG2 134.2 na 90.3 — — — 147.1 −3.55G3 133.5 na 90.8 — — — 148.1 −3.67U4 138.5 100.6 90.9 — — — 161.7 −4.44G5 133.3 na 90.3 — — — 147.4 −3.85C6 137.7 94.7 91.5 — — — — −4.18U7 137.6 101.2 91.4 — — 80.9 — −4.50C8 137.4 94.2 89.3 73.7 — — — −5.14A9 137.3 152.7 85.6 — — — — −2.46G10 — na 81.3 70.9 71.1 — 144.7 −2.72U11 140.7 100.5 — — — — 160.8 −4.32A12 136.3 150.5 90.8 — — — — −4.15C13 138.1 96.9 90.6 72.6 — — — −4.39G14 133.4 na — — — — 144.7 −3.31A15 139.2 151.6 — — — 80.9 — −2.02G16 135.0 na 89.0 — — 80.7 — −3.01A17 138.2 153.0 89.2 — 71.1 78.2 — −4.76G18 134.2 na 90.6 — 71.5 — 146.6 −2.66G19 132.2 na 87.5 — — — 145.1 −4.32A20 136.6 152.0 91.4 72.1 — 80.1 — −4.39A21 135.7 152.0 — — — — — −3.40C22 136.6* 95.1 90.8 — — — −4.23C23 137.4* 96.6 — — — — −3.39G24 134.0 na — — — — 148.6 −3.45C25 138.5 94.5 91.3† — — — — −4.36A26 136.8 150.8 90.3 — — — — −3.92C27 138.1 94.6 91.2 — — — — −4.27C28 138.9 95.0 91.7† — — na — −4.47C29 139.3 95.5 (90.3) — — — −4.18

na, Not applicable.*, † Overlapped in proton dimension.Chemical shifts are given in p.p.m. The 31P and 13C data were obtained at 303 K. The 15N data

were measured at 283 K. The chemical shift standards used are described in Materials andMethods. 13C and 15N chemical shifts are accurate to about 20.05 p.p.m. 31P chemical shifts areaccurate to about 20.03 p.p.m.

is that U11H(6) and A9H(1'), and A9H(8) and U11H(1')

give NOEs to each other because the ‘‘underside’’ ofthe ribose of A9 faces the underside of the ribose ofU11. The G10 bulge must be associated with a kinkin the backbone of E73.

Model building

On the basis of the analysis just described, a totalof 80 starting models for E73 were calculated bydistance geometry and simulated annealing, as

Figure 9. Base-pairings in E73from A9 to A20.

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Figure 10. Stereo view of a refinedmodel of E73. This view is color-coded so that the 3 regions of themolecule can be distinguished. Thestem is blue (G1 to C6, G24 to C29),the connection is black (U7 to C8, C22to C23), and the loop is red (A9 toA21).

described in Materials and Methods. The input dataincluded 212 NOE-based distance restraints, 68hydrogen-bond distances, 162 dihedral restraints,and 13 base-pair planarity restraints. The dihedralrestraints were of three kinds: (1) 58 ribose puckerangles and x angles estimated directly fromexperimental data, (2) 40 a and z angles set on thebasis of 31P chemical shifts, and (3) 57 b, g and e anglesinferred less directly to be A form-like (see Materialsand Methods).

The success rate was remarkably poor. Of the 80refined, distance geometry models computed, 76 (!)were rejected on the grounds of high total energy andmultiple violations of NMR restraints. Only fourwere satisfactory by both criteria: low energy and noviolations of experimental restraints. (No model wasfound that had low energy and multiple restraintviolations, or high energy and no restraintviolations.) These models all had the same topologyas the semi-quantitative model published earlier,and had acceptable bond lengths and bond angleseverywhere (Szewczak et al., 1993b). The all-atom,root-mean-square difference (r.m.s.d.) between thefour distance geometry models in the regionbetween A9 and A21 was 1.23 A.

The four distance geometry models were refinedindependently five times each, using the fullrelaxation matrix method outlined in Materials andMethods. The restraints were: 181 distances relatedto NOEs involving mainly exchangeable protons,volumes measured for 133 different non-exchange-able proton NOE crosspeaks (at three mixing timeseach), 154 dihedral angles and 13 planarity restraints.As it turned out, the initial distance geometry modelsfit the NOE intensity data quite well; starting Rfactors for NOE intensities were about 8%, andrefinement lowered the R factor only to about 6%. Sixrefined models emerged from three of the fourstarting structures that had both low total energy anda small number of violations of restraints. Thediscussion that follows is based on that set of models.

The final models

Figure 10 shows one of the fully refined modelsthat emerged from this study. For the purposes of thediscussion that follows, E73 is divided into threeparts: a stem (G1 to C6, G24 to C29; the blue regionin Figure 10), a connection region (U7–C8, C22–C23;the black region in Figure 10), and a loop (A9 to A21;

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Figure 11. Superposition of E73 loop structures. The 6 best final models for E73 are shown here, superimposed overresidues A9 to A21. Hydrogen atoms are not shown to improve clarity. The Watson-Crick pair G18:C13 is visible in thecenter. Average pairwise r.m.s.d. in this region is approx. 1.11 A.

the red region in Figure 10). The stem is A-formdouble helix, as its sequence and all the dataindicated that it had to be. The stem regions of thesix final models superimpose with an all-atom,r.m.s.d. of 0.6 A. The loop is almost as well-determined; the all-atom, r.m.s.d. in the loop regionis 1.11 A (Figure 11).

Because of a lack of constraints, there was alarge variation in the conformation of theconnection region from one model to thenext, and since the conformation of the connectionregion determined the relative position of thestem and the loop, the angle between the axesof these two regions varied a lot: by 230° arounda mean of about 0°. It is possible that the connectionhas a well-defined conformation in solution, and thatthe relationship between the stem and the loop isfixed, and it is likely that the pyrimidine-pyrimidinejuxtapositions in the connection may play animportant role in marrying the non-canonical A-Apair at the bottom of the stem with the Watson-Crickbase-pair at the top of the stem. The problem is thatthe data are not good enough.

Discussion

Model building

In retrospect, the difficulty we experienced inderiving models for E73 is not surprising. A nucleicacid’s conformation is determined by the sixbackbone torsion angles and the glycosidic torsionangle of each of its nucleotides. The number of NOEs,sugar pucker angles and glycosidic torsion anglesmeasured in E73 significantly exceeded seven pernucleotide, but because of the way the data weredistributed in the molecule, some backbone torsion

angles were hardly determined at all. For that reason,many distance geometry models had conformationalfaults that could not be rectified by simulatedannealing. Fortunately, all such models could beidentified on the basis of their high total energies,their poor covalent geometries, and their numerousviolations of experimental restraints.

The reader will note that considerable use wasmade of constraints based on 31P chemical shifts. It iswidely held that 31P chemical shifts in nucleic acidsreflect a and z angles (Gorenstein, 1984), and thus onecan argue that it is reasonable to hold E73 a and zangles to A-form values for those phosphate groupswhose 31P chemical shifts are in the A-form range,which we did. A case can also be made forrestraining b, g and e in the stem region. Not only are31P chemical shifts ‘‘normal’’ in that part of themolecule, the NOEs observed correspond exactly towhat is anticipated for A-form double helix. Theapplication of such restraints in the connection andthe loop, as was done for residues 12, 13, 19, 20, 21,22 and 23 is harder to justify.

Our first model building computations were doneomitting all torsion angle restraints based on 31Pchemical shifts, and nothing acceptable emergedafter a reasonably large number of independenttrials. Apparently, the odds of finding a refinablemodel within the universe of distance geometrymodels possible in the absence of these restraintswere too low. Thus the rationale for including31P-derived torsional restraints was heuristic; satis-factory models were obtained only when they wereincluded.

Because restraints were used that cannot bedirectly justified experimentally, we do not argue thatwe have uniquely and unambiguously determinedthe conformation of E73 in solution. What we have

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done instead is to find conformations for the E73sequence that are A-like, except where the datarequire otherwise. The observation that low-energymodels consistent with the data and the covalentstructure of E73 were obtained this way lends themplausibility, but we cannot exclude the possibilitythat some other, less A-like conformation is in fact thetrue conformation of E73 in solution. We do maintain,however, that the base-pairing and topology of thatless A-like conformation of E73, if it exists, must bethe same as those of the models displayed here.

Finally, we question the value of the full matrixrelaxation computations we did. As pointed outearlier, the improvement gained in the fit of NOEintensity data to models was modest, as was theimprovement in the convergence of the distancegeometry models: the r.m.s.d. for the initial set ofdistance geometry models was 1.23 A (loop only),while the corresponding r.m.s.d. in the final modelset was still 1.11 A. In fact, the semiquantitativedistance geometry model published in 1993(Szewczak et al., 1993b) would be an acceptablemember of the final model set; its loop geometry hasan all-atom r.m.s.d. relative to the final set of 1.24 A.

Additional support for the conformation pro-posed for the SRL

The credibility of the E73 model that has emergedis supported by its capacity to rationalize spectro-scopic observations that were not used to derive it.For example, the H(1') protons of C13 and G18 bothresonate about 1 p.p.m. upfield of the normal regionfor H(1') protons. The model places both of themdirectly over adenine rings, where strong, upfieldring current shifting should occur. The H(8) of A9 isalso upfield shifted; it lies over the base of C8. TheH(2) of A12, on the other hand, resonates welldownfield of all the other AH(2) protons in the E73spectrum because, unlike them, it has no neighbor-ing base oriented so as to shift it upfield. Thedownfield positions of the resonances of A17H(1') andseveral H(3') and H(2') protons reflect their placementnear the edges of neighboring bases.

E73’s relaxation properties also make structuralsense. The H(8)-C(8)

1H-13C HMQC crosspeak for A15is the sharpest in the HMQC spectrum of E73. A15is the penultimate adenine residue in the loop, andis not hydrogen-bonded to any other residue. It oughtto be the most mobile base in the molecule andshould have the C(8) with the longest carbonrelaxation time. The two protons that have the longestt1 relaxation times (by far) are the H(2) protons of A17and A12 (data not shown). A12 and A17 participatein side-by-side pairings with G residues, whichisolates their H(2) protons from neighboring protonsmuch more effectively than would be the case if theyparticipated in Watson-Crick AU pairs. The H(8) ofA17 (at 8.10 p.p.m.), which has more protonneighbors than purine H(8) protons in a helical nucleicacid generally (see above), is one of the more quicklyrelaxing aromatic protons.

The imino proton resonances of G10, G14 and G19are all observable even though none of them ishydrogen-bonded to another base. In addition, theimino proton faces of all three are resistant to attackby kethoxal even though their pairings apparentlyleave them exposed (Noller, H. F. and Moazed, D.,personal communication). The structure againsuggests why they are protected. The imino proton ofG10 is about 2 A from the phosphate oxygen atom ofA20, which suggests that a hydrogen bond forms.Only modest alteration in the conformation of themolecule would bring the imino proton of G19 intothe same relationship with respect to the phosphateoxygen atoms of A12. The imino proton of G14 isfurther from the nearest phosphate oxygen atom(A17) than that, but the possibility of a water-mediated hydrogen bond deserves consideration(SantaLucia et al., 1992).

Comparisons with other RNA structures

The sarcin/ricin loop is clearly not a 17 base loop atall. It is instead a compact hairpin structure with onlya dinucleotide loop. The abundance of unconven-tional base-pairings, the presence of a bulged base,and the absence of sequence variation in the SRL inall 23 S-like rRNAs made it impossible to diagnoseits conformation from sequence data; it was a loopfaut de mieux. Many other RNA sequences that arenow classified as loops will likely turn out to bestructured in equally idiosyncratic ways.

The SRL is an assembly of three smaller motifs,which have been characterized in other contexts: aGNRA tetraloop, a bulged G motif, and a shortA-type helix. Evidence for the similarity between thetop of the SRL and tetraloops of the GNRA class maybe found in the observation that proton andphosphorus nuclei in analogous residues havesimilar chemical shifts, and the same unusual 1H-1HNOEs are observed (Heus & Pardi, 1991). Forexample, the H(1') resonance of G18 is upfield-shiftedin both molecules, and both exhibit a ‘‘backwards’’NOE between G18H(1') and A17H(8). E73 even showsthe same broadened 31P resonance for G14pA16 asGAAA tetraloops do in other RNA molecules(Legault & Pardi, 1994). This resonance may be inintermediate exchange due to conformational flexi-bility within the GNRA tetraloop (P. Legault and A.Pardi, personal communication).

Sequences identical with the A9 to A12 and G19 toA21 sequence, which form the bulged G motif in E73,are widespread in RNAs. It occurs in loop E ofeukaryotic 5 S rRNA, for example, and in the hairpinribozyme (Butcher & Burke, 1994). There are two ofthem in loop E of procaryotic 5 S RNAs (Specht et al.,1990). The conformation of loop E from Xenopus 5 SrRNA was independently determined by Tinoco andco-workers about the same time we solved E73(Wimberly et al., 1993). Where the sequences of thetwo RNAs are the same, corresponding protons havenearly identical proton chemical shifts, the 31Pspectrum of loop E has outlying resonances that

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correspond to those assigned to pC8, pA9 and pG10in E73, and the same distinctive interproton NOEsare seen. Not surprisingly, the model proposed forloop E closely resembles the one we arrived at for theA9 to A12, G19 to A21 region of E73. For some reason,the Tinoco group did not detect the imino protonresonance of the loop E equivalent of G10, andconsequently could not observe the G10H(1) NOEs toG19H(2') and G19H(3') we saw and that place thatresidue unambiguously.

It is clear that RNA motifs have spectroscopic‘‘fingerprints’’. When sequences are studied in thefuture that might give rise to a GNRA tetraloop or abulged G motif, for example, a lot of time could besaved by looking for the features described above sothat a conformational diagnosis can be made as soonas possible. Had we been aware of this possibility,and had the loop E structure been available to us, ourprogress might have been accelerated by a wholeyear.

Finally, it has long been accepted in the RNAcommunity, almost as an article of faith, that RNAsare modular, that the stem/loop structures soabundant in RNAs are stable domains, whoseconformations are independent of context. Theconformation of E73 suggests that modularity caneven be found within stem/loop structures. It nowappears that small RNA secondary structureelements can be concatenated to make stablestem/loops.

On the relationship of the conformation of theisolated oligonucleotide to that of the SRL in theribosome

Is the model for E73 just presented relevant to theconformation of the SRL in the ribosome? Beyond theobservation that both the SRL in the ribosome andE73 (in vitro) are substrates for ricin and a-sarcin, theonly evidence available comes from the chemicalprotection experiments of Noller and co-workers(H. Noller, T. Gabriel, and D. Moazed, unpublishedresults). They have probed the Watson-Crickhydrogen-bonding faces of SRL G, A and C residuesin 23 S rRNA from E. coli, which is identical insequence with the SRL from rat (i.e. E73) from A9 toA20. The only bases whose hydrogen-bonding facesare fully reactive are A12, A15, G16, A17 and A20,which is what you would predict from thebase-pairing found in E73, granted that G10, G14 andG19 are all unreactive, as discussed earlier. Thus itis likely that the conformation of the SRL is the samein 23 S rRNA as it is in E73.

The Noller group has also probed 50 S ribosomalsubunits from E. coli where the SRL is more protectedthan it is in naked 23 S rRNA, but where nothing thatis protected in naked 23 S rRNA is more reactive.Thus, while the possibility that the conformation ofthe SRL in the ribosome is not the same as it is 23 SrRNA and E73 cannot be ruled out, it is possible thatthey are the same, and that the SRL gains additionalprotection in the ribosome from its interactions withneighboring ribosomal components.

Cytotoxin recognition of the SRL

Wool and his colleagues have identified theresidues in the sarcin/ricin loop that are essential forits activity as a substrate for ricin and a-sarcinby mutagenesis techniques (Endo et al., 1990;Gluck et al., 1992; Wool et al., 1992). They haveshown that ricin will depurinate the appropriateA residue of any GAGA tetraloop that is closed bya short, Watson-Crick helix. Thus ricin recognizesthe top of the sarcin/ricin loop only, a conclusionfully consistent with recent model-buildingstudies (Monzingo & Robertus, 1992; S. Mean,K. Morris, H. Noller, and I. Wool, personalcommunication).

Even though a-sarcin and ricin attack adjacentresidues, a-sarcin seems to recognize a different partthe SRL. Mutants that abolish sarcin recognition arefound below C13:G18, the base-pair that closes thetetraloop. Residue G10 seems to be particularlyimportant. Deletion of that guanine residue, orits substitution by any other base abolishesrecognition by a-sarcin, even though G10 is sixresidues away from G16 (A. Gluck & I. G. Wool,personal communication). How does a small enzymelike a-sarcin recognize G10 but cleave at G16,even when the top of the loop is disrupted by amismatch?

Two explanations suggest themselves. First,a-sarcin may recognize the SRL in a completelydifferent conformation from that described here.This hypothesis is not implausible, given thatthe loop itself is not very stable thermodynamic-ally (Szewczak et al., 1991), and it would beconsistent with speculation that conformationalchange in the SRL is coupled to translocation(see below). A second, simpler explanation issuggested by the concentration-dependence ofa-sarcin’s specificity. At low concentrations(below 30 nM) a-sarcin is quite specific, cleavingonly one bond in ribosomal RNA, but at higherconcentrations it cleaves on the 3' side of Gresidues non-specifically (Wool, 1984). Perhapsthe enzyme’s binding site for RNA has two parts,a part with a generalized, but low affinity forRNA that promotes G-specific cleavage, and asecond part that recognizes bulged G motifs withhigh affinity. As Figure 12 shows, G10, the bulged G,is on the same side of the loop as the phosphate groupof the diester that is cleaved, and is only 15 Aaway from it. Furthermore, the immediate context ofthe bond that is cleaved is A-form-like in itsconformation.

It is intriguing that the bulged G motif in loop Eof eukaryotic 5 S RNA interacts with both L5and transcription factor IIIa (Romaniuk, 1989;Allison et al., 1991), and that the bulged G motifin the SRL interacts with elongation factors(Moazed et al., 1988) as well as with a-sarcin (A.Gluck, & I. G. Wool, personal communication). Itwould not be surprising if the bulged G motif playsa role in the recognition of RNAs by proteinsgenerally.

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Figure 12. The a-sarcin binding surface of the SRL. E73is shown rotated so that the surface that interacts witha-sarcin is in view. The phosphate group attacked bya-sarcin (G16pA17) is yellow. G10 is yellow also.

Materials and Methods

RNA

All E73 samples were prepared by Y.-L. Chan and Ira G.Wool of the University of Chicago using bacteriophage T7RNA polymerase and synthetic DNA templates (Szewczaket al., 1991). d5-CTP was prepared by bisulfite-catalyzedexchange using 2H2O as the deuterium source (Brush et al.,1988; Hayatsu, 1976), and d8-GTP was prepared byuncatalyzed exchange with 2H2O (Benevides et al., 1984).E73-S (see Figure 1) was synthesized chemically by the YaleUniversity School of Medicine Protein and Nucleic AcidChemistry Facility, and deprotected as described byWebster et al. (1991). Following dialysis against 50 mMKCl, 15 mM NaCl, 0.5 mM EDTA, 10 mM phosphate (pH7.6), samples of both E73 and E73-S were brought toconcentrations between 1 and 3 mM by ultrafiltration.

Data acquisition

Routine one and two-dimensional proton spectra wereacquired using the Yale 490 MHz spectrometer. Three-dimensional homonuclear experiments, experiments in-volving 31P, and homonuclear experiments requiringcomposite mixing pulses were implemented on a BrukerAM-500 spectrometer using an inverse detection probewith a broadband X-channel. Heteronuclear 1H-13C and1H-15N correlation experiments, and gradient-enhancedexperiments were done on a General Electric Omega-500PSG spectrometer. NMR data were processed off-lineusing Felix 2.0. Most spectra were apodized using a90°-shifted sine-squared window function. Unless other-wise specified, all contour plots are drawn using geometricspacing between contours.

Dioxane was used as the chemical shift standard(3.741 p.p.m.) for proton experiments, while an externaltrimethyl phosphate standard (0.0 p.p.m.) was usedto reference all 31P spectra. The default GE spectrometer13C and 15N references were used as the chemicalshift standards for other heteronuclear correlationexperiments.

Imino proton spectroscopy

One-dimensional imino proton spectra were collectedusing the twin pulse method (Kime & Moore, 1983). Theoffset was placed near 15 p.p.m. and a sweep width ofabout 14,000 Hz was used. The total recycle time was 0.5to 0.8 second. Imino proton difference NOEs werecollected the same way, with presaturation of the desiredfrequency for 300 milliseconds. Decoupler power was setto a level that gave approximately 80% saturation of theirradiated peak. The total recycle time for each scan wasapproximately 2.0 seconds. On and off-resonance spectrawere collected in an interleaved fashion.

A self-refocused 1-3-3-1 (SR-1331) solvent suppressionpulse sequence (Takegoshi et al., 1990) with TPPI phasecycling (Marion & Wuthrich, 1983) was used to collecttwo-dimensional NOESY spectra of exchangeable protons.A 40 millisecond homospoil pulse was included during themixing time to dephase residual water magnetization.Random variation of the mixing time was included in theseexperiments, as in all other NOE experiments, to suppresszero quantum correlations (Macura et al., 1981). Recycletimes of two seconds or longer were used for these and allother experiments in which observation of both non-exchangeable and exchangeable nuclei was desired.

The SRL in translation

As pointed out earlier, elongation factor binding tothe ribosome protects several SRL residues in E. coliribosomes from chemical modification: residuescorresponding to G10, A15 and G16 (both EF-G andEF-Tu) and A20 (EF-G only: Moazed et al., 1988). Thisis consistent with a model for elongation in which thesole function of the SRL is to serve as the bindingsite for both factors. Depurination of A15 andcleavage of the G16-A17 phosphodiester bond wouldbe pictured as inhibiting translation by weakeningthe interaction of the ribosome with elongationfactors.

Alternatively, the sarcin/ricin loop may play anactive role in elongation. Changes in SRL confor-mational or changes in the way the SRL interacts withother parts of the ribosome may trigger the changein ribosome conformation that occurs duringelongation (Wool et al., 1992). For example, if the SRLtetraloop had to make a tertiary interaction withanother part of 16 S or 23 S rRNA at some phase inthe elongation cycle, as tetraloops can do (Murphy &Cech, 1994; Michel & Westhof, 1990), covalentdamage to the tetraloop would be devastating. Inmodels of this class, elongation factors catalyze theSRL conformational change required, and theireffects on the chemical reactivity of the SRL can beeither direct or indirect. It would be interestingto know whether ricin and a-sarcin-treated ribo-somes retain the capacity to carry out factor-freetranslation.

If the putative conformational change involves thesecondary structure of the SRL, then a conformationfor the SRL must exist that is different from thatdescribed here. If the conformational switch inquestion occurs at the tertiary or quaternary level,the SRL need have only one conformation in theribosome, presumably that described above.

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Non-exchangeable proton spectroscopy

Non-exchangeable proton spectra were acquired withrecycle times of 2.5 to 5.0 seconds, with the shorterdelays (12 seconds) being used for spectra intended forassignment purposes, and longer delays (15 seconds)being used to measure the buildup of NOE intensities.

Magnitude COSY spectra (Aue et al., 1976; Nagayamaet al., 1980) were collected with presaturation to attenuatethe residual 1H2HO peak. The decoupler and transmitterfrequencies were synchronized using a cable splitter(Zagorski, 1990). Phase-sensitive DQF-COSY spectra(Wokaun & Ernst, 1977; Bodenhausen, 1981) were collectedusing States phase cycling (States et al., 1982). H(1')-H(2')

coupling constants were determined by the method of Kim& Prestegard (1989), which corrects for peak cancellationeffects by using the separation of extrema in bothabsorptive and dispersive phasing of the crosspeaks tocalculate scalar coupling constants.

A clean TOCSY (Braunschweiler & Ernst, 1983; Bax &Davis, 1985; Griesinger et al., 1988) experiment with TPPIphase cycling (Marion & Wuthrich, 1983) was used toidentify ribose spin systems. Experiments with severalTOCSY mixing times between 50 and 150 millisecondswere collected.

Non-exchangeable proton NOESY spectra (Jeener et al.,1979) were collected using States phase cycling (States et al.,1982). NOE buildup NOESY spectra were collected with50, 100, and 150 milliseconds mixing times.

Heteronuclear spectroscopy

A gradient-enhanced, jump-return, spin-echo pulsesequence (Szewczak et al., 1993a) was used to collect aproton detected, natural abundance 1H-15N correlationspectrum of E73 at 10°C. In order to achieve maximumresolution in the 13C dimension, natural abundance,proton-detected 1H-13C HMQC spectra (Varani & Tinoco,1991a) were collected using a 13C sweep width of 6500 Hz,with the offset set at 80 p.p.m. so that H(2), H(8) and H(6)

chemical shifts would be aliased into a region of thespectrum that does not overlap with the other 1H-13Ccrosspeaks. The experiment was optimized forJXH = 200 Hz, and 8 kHz GARP decoupling (Shaka et al.,1985) was applied during acquisition to collapse peakmultiplets in the proton dimension.

One-dimensional 31P spectra were collected using asimple pulse-acquire experiment with 5000 Hz broadband1H decoupling during acquisition. Except for the initial,undecoupled 10,000 Hz spectrum used to identify the 5' a,b and g phosphate resonances, 31P sweep widths were 1000to 2000 Hz for E73. Proton detected 1H-31P heteronuclearCOSY experiments (Sklenar et al., 1986; Sklenar & Bax,1987) were collected in inverse mode with low pass anddeuterium filters included in the X-channel. Hetero-TOCSY-based experiments (Kellogg & Schweitzer, 1993;Kellogg, 1992) used a 31P sweep width of 1000 Hz with theoffset at − 3.7 p.p.m. The proton sweep width was 4000 Hzand the proton offset was always set to the 1H2HOfrequency (4.7 p.p.m.). HeteroTOCSY and NOESY mixingtimes were of the order of 40 and 500 milliseconds,respectively, and TPPI phase cycling was used (Marion &Wuthrich, 1983).

Three-dimensional experiments

Three-dimensional heteroTOCSY experiments weredone using only eight scans per t3 FID, with a 31P dimension

(t1) limited to about 80 points, and an indirectly detected1H dimension (t2) of 128 points. The t3

1H sweep width forheteroTOCSY-NOESY and hetero-homoTOCSY exper-iments was 4000 Hz and 2000 Hz, respectively, and for allthree-dimensional experiments the t2

1H dimension was2000 Hz. The heteroTOCSY and NOESY mixing times usedwere similar to their two-dimensional values, andhomonuclear TOCSY mixing times were approximately 80milliseconds.

For the three-dimensional homonuclear 1H cleanTOCSY-NOESY experiment, t3, t1, and t2 were 512, 92 and128 points, respectively, and the proton sweep widthswere all 4000 Hz. For this experiment, the homonuclearTOCSY and NOESY mixing times were 100 and 300milliseconds.

Distance geometry model building

All conformational calculations were done usingX-PLOR, version 3.1 (Brunger, 1992). Protocols were testedusing a standard A-form double helix as a model to ensurethat convergence would be achieved. Base-pairs weredefined by specifying distances between hydrogen-bonddonor and acceptor atoms. For each donor-acceptor pairA-H. . . .B, the A to B and H to B distances were definedusing published distances for Watson-Crick pairs (Saenger,1984), and 2.0(20.3) A for the hydrogen-bond distance and3.02(0.3) A for the heavy-atom distance for non-Watson-Crick pairs. Base-pair planarity was maintained byspecifying a set of six atoms, three from each base, andapplying a 50 kcal mol−1 A−2 penalty for deviating atoms(Brunger, 1992).

Ribose conformations (%N) were determined from JH1'H2'

values using the equation (van den Hoogen, 1988):%N = 114.9 − 14.5(JH1'H2'). An average ribose pucker phaseangle was then calculated, assuming a two-state modelwhere tm = tS = tN = 37°, PS = 162°, and PN = 9°. Individualvalues for the ring dihedrals n0 to n4 were extracted usingPavg and tm in the following equation (Altona &Sundaralingam, 1972): nj = tm cos[P + 4p (j − 2)/5]j = 0, 1, 2, 3, 4. The chi angles were all set to the anticonformation (200(230)°), except for G10, A15 and A17,for which chi was set to 200(260)° because of theirsomewhat stronger intraresidue H(1')-H(8) NOEs.

For the purposes of calculating initial, distancegeometry structures, interproton NOE crosspeaks wereclassified into three overlapping distance ranges: short (1.8to 3.0 A), medium (2.0 to 4.0 A) and long (3.0 to 6.0 A) asfollows. If a crosspeak was of medium or strong intensityat 50 milliseconds it was classified as a short distance. If acrosspeak was weak or not observed at 50 milliseconds, butwas of medium to strong intensity at 100 milliseconds, itwas classified as a medium distance. Long distancescorresponded to the NOEs that were weak, but visible at100 milliseconds. Sequential H(1')-H(8) or H(1')-H(6) distances,which are known to appear ‘‘short’’ because of spindiffusion, were classified as medium. NOEs involvingexchangeable protons were taken as corresponding to adistance of 3.8(21.2) A if they were present in a 10°C 300milliseconds NOESY spectrum.

Loosely bounded dihedral restraints were added for a, b,g, e and z, for every phosphate group having a 31P chemicalshift in the A-form range. In the stem region, each dihedralrestraint was limited to 215° of the A-form value, whilein the loop each angle was allowed to vary by 230°. Norestraint was included for phosphate groups having 31Pchemical shifts outside the A-form range.

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Restrained molecular dynamics calculations

Although usually correct topologically, the coordinatesets that emerge from distance geometry calculations areoften unrecognizable as a molecular structure and must beenergy-minimized before any use can be made of them.This was done using a combination of conjugate gradientminimization, and simulated annealing.

The initial phase of the refinement was done using ashort non-bonded interaction cutoff (4.5 A), a van derWaals term that was repulsive only, no electrostatic term,and no explicit hydrogen-bonding term. After some initialconjugate gradient minimization to idealize covalentgeometries, each structure was heated to 2000 K and theNMR-derived distance, torsional angle, and hydrogen-bonding restraints were slowly increased as the moleculardynamics proceeded. Simultaneously, bond, angle andimproper energy terms were included. After equilibratingthe molecule for six picoseconds at high temperature, arepulsive van der Waals term was turned on, and eachstructure was slowly cooled to 100 K, and then minimized.Molecular dynamics time-steps were of the order of onefemtosecond.

Full matrix refinement

Distance geometry starting structures were furtherrefined using a simulated annealing protocol that includedfull matrix relaxation back calculations to optimize the fitto non-exchangeable proton NOEs (Nilges et al., 1991;White et al., 1992). r1/6 weighting was used for the analysisof NOE crosspeak volumes. Crosspeak volumes weremeasured in non-exchangeable proton NOESY spectracollected at mixing times of 50, 100 and 150 milliseconds,using Felix 2.1. These data replaced most of thenon-exchangeable proton-proton distances used in calcu-lating distance geometry starting models. An isotropiccorrelation time of three nanoseconds, which was obtainedfrom a minimum R-factor grid search that used pyrimidineH(5)-H(6) crosspeaks as a standard, was used throughout. Inaddition, A-form dihedral restraints were relaxed forresidues adjacent to nucleotides having unusual 31Pchemical shifts, and a full Lennard-Jones potential wasused in place of the repulsive-only van der Waals termemployed during distance geometry calculations. Modelswere heated to 1000 K for two picoseconds, slowly cooledto 300 K, and then minimized.

Acknowledgements

We thank Dr Ira Wool for encouraging us to undertakethis project, his help in bringing it to fruition, and keepingus aware of his results on the effects of mutation on the SRL.We also gratefully acknowledge our debt to Yuen-LingChan of the University of Chicago, who prepared the E73samples used in this study. A.A.S. was supported by anNSF predoctoral fellowship. The work was supported bygrants from the National Institutes of Health (AI09167 andGM41651).

ReferencesAllison, L. A., Romaniuk, P. J. & Bakken, A. H. (1991).

RNA-protein interactions of stored 5 S RNA withTFIIIA and ribosomal protein L5 during Xenopusoogenesis. Develop. Biol. 144, 129–144.

Altona, C. & Sundaralingam, M. (1972). Conformationalanalysis of the sugar ring in nucleosides andnucleotides. A new description using the conceptof pseudorotation. J. Amer. Chem. Soc. 94, 8205–8212.

Aue, W. P., Bartholdi, E. & Ernst, R. R. (1976).Two-dimensional spectroscopy. Application tonuclear magnetic resonance. J. Chem. Phys. 64,2229–2246.

Bax, A. & Davis, D. G. (1985). MLEV-17 basedtwo-dimensional homonuclear magnetization trans-fer spectroscopy. J. Magn. Reson. 61, 306–320.

Benevides, J. M., Lemeur, D. & Thomas, G. J., Jr(1984). Molecular conformation and 8-CH exchangerates of purine ribo- and deoxyribonucleotides:investigation by Raman spectroscopy. Biopolymers, 23,1011–1024.

Bodenhausen, G. (1981). Multiple quantum NMR. Progr.NMR Spectrosc. 14, 137–173.

Braunschweiler, L. & Ernst, R. R. (1983). Coherencetransfer by isotropic mixing: application toproton correlation spectroscopy. J. Magn. Reson. 53,521–528.

Brunger, A. (1992). X-PLOR Version 3.1. System for X-rayCrystallography and NMR. Yale University Press, NewHaven, CT.

Brush, C. K., Stone, M. K. & Harris, T. M. (1988). Selectivereversible deuteriation of oligonucleotides: simplifica-tion of two-dimensional nuclear Overhauser effectNMR and spectral assignment of a non-self-comp-lementary dodecamer complex. Biochemistry, 27,115–122.

Butcher, S. E. & Burke, J. M. (1994). A photo-cross-linkabletertiary stucture motif found in functionallydistinct RNA molecules is essential for catalyticfunction of the hairpin ribozyme. Biochemistry, 33,992–999.

Endo, Y. & Wool, I. G. (1982). The site of action of alphasarcin on eukaryotic ribosomes. J. Biol. Chem. 257,9054–9060.

Endo, Y., Mitsui, M., Motizuki, M. & Tsurugi, K. (1987).The mechanism of action of ricin and related toxiclectins on eukaryotic ribosomes. The site and thecharacteristics of the modification in 28 S ribosomalRNA caused by the toxins. J. Biol. Chem. 262,5908–5912.

Endo, Y., Gluck, A., Chan, Y., Tsurugi, K. & Wool, I. G.(1990). RNA-protein interaction. An analysis withRNA oligonucleotides of the recognition by alpha-sarcin of a ribosomal domain critical for function.J. Biol. Chem. 265, 2216–2222.

Endo, Y., Gluck, A. & Wool, I. G. (1991). Ribosomal RNAIdentity Elements for Ricin A-chain Recognition andCatalysis. J. Mol. Biol. 221, 193–207.

Gluck, A., Endo, Y. & Wool, I. G. (1992). Ribosomal RNAidentity elements for ricin A-chain recognition andcatalysis. J. Mol. Biol. 226, 411–424.

Gorenstein, D. G. (1984). Phosphorus -31 NMR. Principlesand Applications. Academic Press, Inc., New York.

Griesinger, C., Ottings, G., Wuthrich, K. & Ernst,R. R. (1988). Clean TOCSY for 1H spin systemidentification in macromolecules. J. Amer. Chem. Soc.110, 7870–7872.

Gutell, R. R., Schnare, M. & Gray, M. (1992). A compilationof large subunit (23 S- and 28 S-like) ribosomalRNA structures. Nucl. Acids Res. 20(suppl.), 2095–2109.

Hausner, T.-P., Atmadja, J. & Nierhaus, K. H. (1987).Evidence that the G2661 region of 23 S rRNA is located

JMB—MS 326


at the ribosomal binding sites of both elongationfactors. Biochimie, 69, 911–923.

Hayatsu, H. (1976). Bisulfite modification of nucleic acidsand their constituents. Prog. Nucl. Acid Res. Mol. Biol.16, 75–124.

Heus, H. & Pardi, A. (1991). Structural features that giverise to the unusual stability of RNA hairpinscontaining GNRA loops. Science, 253, 191–194.

Jeener, J., Meier, B. H., Bachmann, P. & Ernst, R. R. (1979).A two-dimensional nuclear overhauser experimentwith pure phase absorption in four quadrants. J. Chem.Phys. 71, 4546–4553.

Kellogg, G. K. & Schweitzer, B. I. (1993). Two- andthree-dimensional 31P-driven NMR procedures forcomplete assignment of backbone resonances inoligodeoxyribonucleotides. J. Biomol. NMR 3, 577–595.

Kellogg, G. W. (1992). Proton-detected hetero-TOCSYexperiments with application to nucleic acids. J. Magn.Reson. 98, 176–182.

Kim, Y. & Prestegard, J. H. (1989). Measurement of vicinalcouplings from crosspeaks in COSY spectra. J. Magn.Reson. 84, 9–13.

Kime, M. J., Moore, P. B. (1983). Physical evidence for adomain structure in Escherichia coli 5 S RNA. FEBSLetters, 153, 199–203.

LeMaster, D. M. & Richards, F. M. (1988). NMR sequentialassignment of Escherichia coli thioredoxin utilizingrandom fractional deuteriation. Biochemistry, 27,142–150.

Legault, P. & Pardi, A. (1994). 31P chemical shift as a probeof structural motifs in RNA. J. Magn. Reson. B 103,82–86.

Macura, S., Huang, Y., Suter, D. & Ernst, R. R. (1981).Two-dimensional correlated spectroscopy (COSY) formeasurement of 1H-1H spin-spin coupling constantsin proteins. J. Magn. Reson. 43, 259–281.

Marion, D. & Wuthrich, K. (1983). Application of phasesensitive two-dimensional correlated spectroscopy formeasurements of 1H-1H spin-spin coupling constantsin proteins. Biochem. Biophys. Res. Commun. 113,967–974.

Michel, F. and Westhof, E. (1990). Modelling of thethree-dimensional architecture of group I catalyticintrons based on comparative sequence analysis. J.Mol. Biol. 216, 585–610.

Moazed, D., Robertson, J. M. & Noller, H. F. (1988).Interaction of elongation factors EF-G and EF-Tu witha conserved loop in 23 S RNA. Nature (London), 334,362–364.

Monzingo, A. F. & Robertus, J. D. (1992). X-ray analysis ofsubstrate analogs in the ricin A chain active site. J. Mol.Biol. 227, 1136–1145.

Murphy, F. L. & Cech, T. R. (1994). GAAA tetraloop and aconserved bulge stabilize tertiary structure of a groupI intron domain. J. Mol. Biol. 236, 49-63

Nagayama, K., Jumar, A., Wuthrich, K. & Ernst, R. R.(1980). Experimental techniques of two-dimensionalcorrelated spectroscopy. J. Magn. Reson. 40, 321–334.

Nilges, M., Habazattl, J., Brunger, A. T. & Holak, T. A.(1991). Relaxation matrix refinement of the solutionstructure of squash trypsin inhibitor. J. Mol. Biol. 219,499–510.

Noller, H. F., Kop, J., Wheaton, V., Brosius, J., Gutell, R. R.,Kopylov, A. M., Dohme, F., Herr, W., Stahl, D. A.,Gupta, R. & Woese, C. R. (1981). Secondary structuremodel for 23 S ribosomal RNA. Nucl. Acids Res. 9,6167–6189.

Romaniuk, P. J. (1989). The role of highly conservedsingle-stranded nucleotides of Xenopus 5 S RNA in

the binding of transcription factor IIIA. Biochemistry,28, 1388–1395.

Saenger, W. (1984). Principles of Nucleic Acid Structure.Springer-Verlag, New York.

SantaLucia, J., Jr., Kierzek, R. & Turner, D. H. (1992).Context-dependence of hydrogen bond free energyrevealed by substitutions in an RNA hairpin. Science,256, 217–219.

Shaka, A. J., Barker, P. B. & Freeman, R. (1985).Computer-optimized decoupling scheme for wide-band applications and low-level operation. J. Magn.Reson. 64, 547–552.

Sklenar, V. & Bax, A. (1987). Measurement of 1H-31Pcoupling constants in double-stranded DNA frag-ments. J. Amer. Chem. Soc. 109, 7525–7526.

Sklenar, V., Miyoshiro, H., Zon, G. & Bax, A. (1986).Assignment of 31P and 1H resonances in oligonucleo-tides by two-dimensional NMR spectroscopy. FEBSLetters 208, 94–98.

Specht, T., Wolters, J. & Erdmann, V. A. (1990).Compilation of 5 S ribosomal RNA and 5 S ribosomalRNA gene sequences. Nucl. Acids. Res. 18(Suppl.),2215–2230.

States, D. J., Haberkorn, R. A. & Ruben, D. J. (1982). Atwo-dimensional nuclear Overhauser experimentwith pure absorption phase in four quadrants. J. Magn.Reson. 48, 286–292.

Szewczak, A. A., Chan, Y.-L., Moore, P. B. & Wool, I. G.(1991). On the conformation of the alpha sarcinstem-loop of 28 S rRNA. Biochimie, 73, 871–877.

Szewczak, A. A., Kellogg, G. W. & Moore, P. B. (1993a).Assignment of NH resonances in nucleic acids usingnatural abundance 15N-1H correlation spectroscopywith spin-echo and gradient pulses. FEBS Letters, 327,261–264.

Szewczak, A. A., Moore, P. B., Chan, Y.-L. & Wool, I. G(1993b). The conformation of the sarcin/ricin loopfrom 28 S ribosomal RNA. Proc. Nat. Acad. Sci., U.S.A.90, 9581–9585.

Takegoshi, K., Tsuda, S. & Hikichi, K. (1990). Practicalimplementation of the self-refocused 1331 solvent-suppression sequence. J. Magn. Reson. 89, 399–405.

Van den Hoogen, F. (1988). NMR studies concerningbase-base interactions in oligonucleotides. PhD thesis,University of Leiden.

Varani, G. & Tinoco, I., Jr (1991a). Carbon Assignments andheteronuclear coupling constants for an RNAoligonucleotide from natural abundance 13C-1Hcorrelated experiments. J. Amer. Chem. Soc. 113,9349–9354.

Varani, G. & Tinoco, I., Jr (1991b). RNA structure and NMRspectroscopy. Quart. Rev. Biophys. 24, 479–532.

Webster, K. R., Shamoo, Y., Konigsberg, W. & Spicer, E. K.(1991). A rapid method for purification of syntheticoligoribonucleotides. BioTechniques, 11, 658–661.

White, S. A., Nilges, M., Brunger, A. & Moore, P. B. (1992).An NMR analysis of Helix I from the 5 S RNA ofE. coli. Biochemistry 31, 1610–1621.

Wimberly, B., Varani, G. & Tinoco, I., Jr (1993). Theconformation of loop E of eucaryotic 5 S ribosomalRNA. Biochemistry, 32, 1078–1087.

Wokaun, A. & Ernst, R. R. (1977). Selective detectionof multiple quantum transitions in NMR bytwo-dimensional spectroscopy. Chem. Phys. Letters, 52,407–412.

Wool, I. G. (1984) The mechanism of action of the cytotoxicnuclease alpha-sarcin and its use to analyze ribosomestructure. Trends. Biochem. Sci. 9, 14–17.

Wool, I. G., Gluck, A. and Endo, Y. (1992) Ribotoxin

JMB—MS 326


recognition of ribosomal RNA and a proposal for themechanism of translocation. Trends. Biochem. Sci. 17,266–269.

Zagorski, M. (1990). Improved frequency selectivity forpresaturation of H2O in 2D NMR spectra of proteins.J. Magn. Reson. 86, 400–405.

Edited by P. E. Wright

(Received 19 September 1994; accepted 14 November 1994)

Note added in proof: Nierhaus and coworkers have shown that ribosomes treated with a-sarcin arecompetent in factor-free translocation (Hausner et al., 1987).

Documents

The Sarcin / Ricin Loop, a Modular RNA€¦ · The Sarcin/Ricin Loop, a Modular RNA A. A. Szewczak and P. B. Moore* Departments of Chemistry The conformation of a 29 base oligonucleotide