Depurination of A4256 in 28 S rRNA by the Ribosome-inactivating Proteins from Barley and Ricin...

J. Mol. Biol. (1996) 259, 81–94

Depurination of A4256 in 28 S rRNA by theRibosome-inactivating Proteins from Barley and RicinResults in Different Ribosome Conformations

Lovisa Holmberg and Odd Nyga ˚rd*

Department of Zoological Ribosomal function in protein synthesis requires dynamic flexibility of theCell Biology ribosomal structure. The two translational inhibitors derived from seeds ofArrhenius Laboratories E5 ricin and barley destroy the dynamic properties of the ribosome byStockholm University selective depurination of A4256 in the phylogenetically conserved

a-sarcin/ricin loop of mouse 28 S rRNA. As the a-sarcin/ricin loop isS-106 91 Stockholm, Swedeninvolved in binding of elongation factors to the ribosome, depurinationblocks the protein synthesis elongation cycle. Depurination by the barleytranslational inhibitor (BTI) mainly effects eEF-1a related functions, whilericin interferes with the interaction of eEF-2 with the ribosome. Analysisof the ribosomal structure after inhibitor treatment shows that theaccessibility of the rRNAs for single-strand-specific chemical modificationwas altered. Reactivity changes were seen in domains I, II and V of 28 SrRNA and in 5 S rRNA. A majority of the reactivity changes were foundin putative functional regions of the rRNAs, such as the regions involvedin peptidyltransferase activity, subunit interaction and in the binding ofelongation factors. Most of the observed structural changes made therRNAs less accessible for chemical modification, suggesting that theribosomal particles became less flexible after inhibitor treatment. More-over, the modification patterns obtained from the two inhibitor-treatedribosomal particles were only partly overlapping, indicating that thestructure of the large ribosomal subunit differed after ricin and BTItreatment.

Surprisingly, depurination in the a-sarcin/ricin loop of 28 S rRNA alsoaffected the structure of the 3' major domain in 18 S rRNA.

7 1996 Academic Press Limited

Keywords: ribosome-inactivating proteins; ribosome structure; rRNA*Corresponding author structure, dynamics and translation

Introduction

The ribosome is a highly flexible structure inwhich the dynamic properties are a prerequisite forfunction (for a review, see Nygard & Nilsson, 1990).Escherichia coli ribosomes reciprocate between acontracted and an extended conformation duringprotein synthesis (Conway & Lipman, 1964) and arethus present in at least two different forms (Hapke& Noll, 1976). The structural differences between

the extended and contracted forms are dependenton variations in the large ribosomal subunit (vanDiggelen & Bosch, 1973; van Diggelen et al., 1973)and it has been suggested that conformationalchanges in the 23 S rRNA are responsible for theoccurrence of the two ribosomal forms (Burmaet al., 1984). The rRNA structures are interconvert-ible in the presence of elongation factor EF-G andGTP, suggesting that the extended and contractedforms of the ribosome, with their specific 23 S rRNAstructures, are related to the pre and post-transloca-tion forms of the large ribosomal subunit (Spirin,1967; Spirin et al., 1987).

The pre-translocation state of the ribosomeinteracts with elongation factor EF-G/eEF-2, whilethe post-translocation form binds EF-Tu/eEF-1a(for a review, see Nygard & Nilsson, 1990).Both elongation factors bind to the ribosome at

Abbreviations used: BTI, Barley translationalinhibitor; CMCT, 1-cyclohexyl-3-(morpholinoethyl)carbodiimide metho-p-toluene sulphonate; DMS,dimethyl sulphate; RIP, ribosome-inactivating protein;eEF-1a and eEF-2, eukaryotic elongation factors 1a and2, respectively; EF-Tu and EF-G, prokaryotic elongationfactors Tu and G, respectively.

0022–2836/96/210081–14 $18.00/0 7 1996 Academic Press Limited

BTI and Ricin Promote Different Ribosome Conformations82

overlapping binding sites. On the large subunit, thephylogenetically conserved a-sarcin/ricin loop in28 S rRNA is of vital importance for the interactionbetween ribosomes and elongation factors (Hausneret al., 1987; Moazed et al., 1988). Ribosome inacti-vating proteins (RIPs) derived from plant seeds,such as ricin from Ricinus communis, and the trans-lational inhibitor from barley (BTI) Hordeum sp.,interfere with the elongation cycle by depurinatingone base in the a-sarcin/ricin loop, A4256 in mouse28 S rRNA (Endo et al., 1987, 1988; Taylor et al.,1994). Despite the fact that both ricin and BTIremove the same base in 28 S rRNA, the two RIPshave different effects on ribosomal function inrabbit reticulocytes (Nilsson et al., 1986; Nilsson& Nygard, 1986). Ricin interferes with the eEF-2-dependent part of the elongation cycle. Thus,ricin-treated ribosomes have reduced ability to formthe pre-translocation type of complex with eEF-2but retain their ability to trigger the eEF-2 andribosome-dependent GTP-hydrolase activity in thepost-translocation state. On the other hand, BTImainly interferes with eEF-1a related functions. Thetwo inhibitors therefore seem to lock the ribosomein a particular state that prevents further pro-gression of the ribosome through the elongationcycle.

Here, we have studied the effects of ricin andBTI-dependent depurination of A4256 on thestructure of the large ribosomal particle byanalysing the conformation of the rRNAs from thetwo types of ribosomal particles using single-strand-specific chemical modification and primerextension. BTI and ricin-treated subunits displayedspecific modification patterns, indicating that ricinand BTI-treated ribosomes have different confor-mations.

Results

The a-sarcin/ricin-loop is suggested to be acompact hairpin structure containing several un-conventional base-pairs and a dinucleotide loop(Szewczak & Moore, 1995). In mouse 28 S rRNA thenucleotides in the suggested loop consist of A4256and G4257. Several fungi and plant-derived transla-tional inhibitors interfere with ribosomal functionvia a direct effect on the a-sarcin/ricin-loop. Thetwo translational inhibitors derived from the seedsof barley (BTI) and castor beans (ricin) blockribosomal progression through the elongation cycleby depurinating 28 S rRNA at position A4256 (Endoet al., 1987, 1988; Taylor et al., 1994). The efficiencyof depurination of isolated 60 S subunits was muchhigher for ricin than for BTI (Figure 1). Thus, at2 mM Mg2+ ricin was approximately 25 times moreefficient than BTI in depurinating A4256 (Figure 1).This is in agreement with previous reports (Nilssonet al., 1986; Taylor et al., 1994). The ability of BTIto depurinate 28 S rRNA was, however, stronglyinfluenced by the magnesium concentration. Asseen in Figure 1, the extent of BTI-dependentdepurination decreased with increasing magnesium

Figure 1. Effect of Mg2+ concentration on ricin andBTI-dependent depurination of A4256 in 28 S rRNA. Fordepurination, 20 pmol of 60 S ribosomal subunits, in afinal volume of 50 ml, were incubated in the presence of50 pmol of BTI (W) or 3 pmol of ricin (Q) for ten minutesat 37°C. Depurination of the 28 S rRNA was monitored byprimer extension and gel electrophoresis as described inMaterials and Methods. The extent of depurination atA4256 was calculated based on the intensities of thebands at A4256 and U4181 (strong natural stop caused bya pseudouridine residue) using a computer-based imageanalysis system (Holmberg et al., 1992).

concentration, while changes in the magnesiumconcentration had no effect on the ricin-catalyseddepurination of A4256. The latter result is inagreement with the observation by Brigotti et al.(1989).

In reticulocyte lysates, BTI and ricin mainlyinterfere with the eEF-1a and eEF-2 mediatedreactions, respectively (Nilsson et al., 1986; Nilsson& Nygard, 1986). As these observations may reflectdifferences in the structure of the ribosomepopulations treated with the two RIPs, we studiedthe conformation of rRNA in BTI and ricin-treated60 S subunits using chemical modification andprimer extension. The 60 S subunits were incubatedat two different concentrations of magnesium, 2 and7 mM, and were then treated with ricin or BTI. Forthe evaluation of the results it was essential thatthe extent of depurination was similar with bothRIPs, irrespective of the Mg2+ concentration used.Therefore, the BTI and ricin concentrations wereincreased to 16 and 0.33 mM, respectively, to obtainmore than 70% depurination in all experiments(Figure 2).

The rRNA in inhibitor-treated and controlribosomes was modified using the single-strand-specific reagents DMS and CMCT. The observedmodification patterns of 5 S, 5.8 S and 28 S rRNA incontrol subunits at 7 mM Mg2+ were identical withthat previously reported (Holmberg et al., 1992,1994b). However, at 2 mM Mg2+ the reactivity ofsome of the nucleotides was changed (unpublishedresults). The translational inhibitors altered thechemical modification pattern in domains I, II andV of 28 rRNA and in 5 S rRNA. Furthermore, the60 S subunits displayed inhibitor-specific structural

BTI and Ricin Promote Different Ribosome Conformations 83

Figure 2. Autoradiogram showingthe extent of BTI and ricin-depen-dent depurination of A4256 in 28 SrRNA from the ribosomal particlesused in the chemical modificationexperiments. The 60 S subunits(0.7 mM) were treated with BTI(16 mM) or ricin (0.33 mM) at theMg2+ concentrations indicated. Afterincubation for five minutes at 37°C,CMCT was added to a final concen-tration of 0, 20 or 100 mM and theincubation continued as describedin Materials and Methods. U, G, Cand A are sequence lanes. The newnatural stops at positions G4268 andG4269 are indicated.

differences and the modification patterns obtainedfrom ricin and BTI-treated ribosomes were onlypartly overlapping (Figures 3 to 5).

As comparable extent of depurination in ricin andBTI-treated ribosomes required considerably higherconcentrations of BTI, it was essential to eliminatefootprinting on the rRNA by ribosome-bound BTIas the primary cause of the reduced chemicalreactivity seen in BTI-treated ribosomes. For thispurpose, subunits were depurinated by ricin andthen allowed to interact with BTI before chemicalmodification. The resulting modification patternwas similar to that seen in the presence of ricin only,suggesting that the BTI-specific reductions inchemical accessibility did not result from footprint-ing by ribosome-bound BTI (one example is shownin Figure 8C). This conclusion is supported by theobservation that ricin and BTI, at the concentrationsused, did not inhibit the ribosome and eEF-2-depen-dent GTP hydrolase activity (data not shown).

Domain I of 28 S rRNA

The degree of BTI and ricin-dependent changes inthe chemical accessibility of the rRNAs wasinfluenced by the Mg2+ concentration despite thefact that the extent of depurination was the same atthe different concentrations of Mg2+ used. At 2 mMMg2+, the region containing helices 19 and 20became more sensitive to chemical modificationwithin both ricin and BTI-treated 60 S subunits(Figures 3A and 6A). This was the only region in28 S rRNA where nucleotides were exposed in asimilar way by the two inhibitors. At 7 mM Mg2+,the RIPs caused considerably less structuralchanges in the rRNAs. Under these conditions,weakly reactive bases in the apical loops of helices21 and 22, and in the bulge between helices 4 and

23, became less susceptible to modification inBTI-treated subunits (Figures 3A and 6B). In theBTI-treated subunits, two nucleotides located inhelix 17 adjacent to the internal bulge bridginghelices 17 and 18 served as natural stops for thereverse transcriptase at both Mg2+ concentrationsused (Figure 3A).

Domain II of 28 S rRNA

Ricin and BTI-treatment of 60 S subunits resultedin complex structural changes in domain II of 28 SrRNA. Both RIPs caused similar effects on thereactivity of bases in the structurally and function-ally conserved region containing hairpins 43 and 44(Raue et al., 1990). Here, ricin and BTI induced anew natural stop at G1777, located in helix 43, atboth concentrations of Mg2+ used. Furthermore, thetwo nucleotides G1808 and U1809, located in helix44, became less accessible for chemical modificationin ribosomes treated with the RIPs at 7 mM Mg2+,although the effect of BTI was more pronouncedthan that observed with ricin (Figures 3A and 7C).

Surprisingly, BTI and ricin caused oppositereactivity changes in two regions of domain II. Theregion around helices 45 and 46 was less susceptibleto chemical modification in BTI-treated than incontrol subunits (Figures 3A and 7B). The effect wasmost clearly seen at nucleotides U2047, U2052,A2061 and A2062 but the whole sequence betweenC2043 and A2067 was more or less protected againstmodification. In contrast, treatment with ricinresulted in increased reactivity of several bases inthis region, although the exposure was most clearlyseen at nucleotides A2101 and A2103 (Figures 3Aand 7B).

The second region that showed opposite reactiv-ity changes involved nucleotides A1375 to A1377,


Figure 3(A) legend opposite


(B)

Figure 3. Secondary structure model of mouse 28 S rRNA (Gutell et al., 1993) summarizing the BTI andricin-dependent changes in the chemical modification pattern of domains I and II (A) and V (B). The data representthe average density of autoradiograms (Holmberg et al., 1992) from independent experiments. Bases exposed to (squares)or protected from (circles) chemical modification in ricin (open symbols) and BTI-treated ribosomal particles (filledsymbols). Reactivities changed by 20 to 40% (small symbols). Bases with reactivities changed by more than 40% (largesymbols). New natural stops (t and T) in ricin and BTI-treated subunits, respectively; P indicates the position ofthe depurination site A4256 in domain VI.

located in the apical loop of helix 31. At 2 mM Mg2+,these bases showed increased exposure to DMSmodification in BTI-treated 60 S subunits butbecame less accessible to modification followingtreatment with ricin (Figures 3A and 7A).

Domain V of 28 S rRNA

Prominent changes in the modification patternbetween control and RIP-treated subunits wereseen in domain V. In the upper part of the domain,


Figure 4. Secondary structure model of mouse 5 S rRNA (Sarge & Maxwell, 1991) showing inhibitor-dependentchanges in the chemical modification pattern. The accessibility changes are indicated by the symbols as in Figure 3.

a higher stability of the RNA was observed in theBTI-treated subunits and six G-C base-pairs locatedon both sides of the loop connecting helices 82 and83 functioned as new natural stops for the reversetranscriptase (Figure 3B). BTI treatment of thesubunits at 2 mM Mg2+ also resulted in the specificexposure of one nucleotide, A3974, located in theapical loop of helix 92 (Figures 3B and 8A). Incontrast, several weakly reactive nucleotides locatedclose to the so-called peptidyltransferase ringbecame less reactive after treatment with BTI. Here,the RNA sequence between helices 93 and 78, thebulge-loop in the latter helix, and the region at thebase of helix 94 became less available formodification than in control subunits at bothconcentrations of Mg2+ used (Figures 3B and 8C).Additional protected nucleotides were found in theweakly exposed helix 96.

Unlike the BTI-induced structural changes,ricin-dependent alterations of the modification

pattern were seen only at 2 mM Mg2+. Inricin-treated 60 S subunits, part of the peptidyl-transferase ring, the bulge-loop of helix 78 and helix96 were protected against modification. Moreover,a specific protection was observed at U4034 (Fig-ures 3B and 8B).

Domain VI of 28 S rRNA

Treatment of 60 S subunits with the twotranslational inhibitors did not affect the chemicalmodification pattern of nucleotides in domain VI.However, both inhibitors induced two strongnatural stops at the base of the a-sarcin/ricin-loop(helix 100) at positions G4268 and G4269 (Figures 2and 3B). The occurrence of additional but muchweaker natural stops in the region was noted. Helix100 is suggested to be in close proximity to helix 43in the three-dimensional structure of the ribosome(Moazed et al., 1988). These two helices contained

Figure 5. Secondary structuremodel of mouse 18 S rRNA (Gutell,1994; Holmberg et al., 1994b) sum-marizing the BTI and ricin-depen-dent reactivity changes. The re-activity changes are indicated by thesymbols as in Figure 3.


Figure 6. The extent of single-strand modification of bases indomain I of 28 S rRNA was affectedby BTI and ricin-treatment of 60 Ssubunits. The subunits were incu-bated as described in the legend toFigure 2.

the only sequences where identical natural stopswere found as a result of BTI and ricin-treatment ofthe 60 S particles.

5 S rRNA

The high degree of secondary structure in 5 SrRNA (approximately half of the nucleotidesare suggested to be involved in Watson–Crickbase-pairing) made 5 S rRNA relatively inaccessibleto chemical modification by the single-strand-specific reagents. Despite this fact, A5 in helix Iwas partly available for DMS-modification in the

control subunits. When the 60 S particles weretreated with BTI this nucleotide became inacces-sible for modification (Figures 4 and 9A), whereasA74, A88 and A90 located in the E and D-loopsbecame more exposed to the reagent (Figure 9B).The effect on the latter three bases was mostpronounced at 2 mM Mg2+. The two bases A88and A90 were also exposed as a result of treatmentwith ricin, although to a lesser extent than in theBTI-treated subunits. In addition, A42 located inthe C-loop was partly shielded from modificationin subunits treated with ricin at 2 mM Mg2+ (Fig-ure 9A).

Figure 7. The reactivity of bases indomain II of 28 S rRNA was affectedby BTI and ricin-treatment of 60 Ssubunits. The subunits were treatedwith BTI or ricin and modified byCMCT as described for Figure 2. ForDMS-modification the samples wereincubated in the presence of 0, 20or 90 mM DMS for five minutes at37°C.


Figure 8. The chemical modifi-cation pattern of domain V in 28 SrRNA was altered after ricin andBTI-treatment of 60 S subunits. Thesubunits were incubated with BTIand ricin as described in the legendto Figure 2. For the combined treat-ment, the ribosomal particles werefirst incubated for five minutesat 37°C in the presence of ricinfollowed by an additional fiveminutes incubation in the presenceof BTI. The subunits were chemi-cally modified as described forFigures 2 and 7.

The 3' major domain of 18 S rRNA

It was previously observed that although ricin hasa specific effect on the 28 S rRNA, many of theresulting biochemical characteristics of the depuri-nated 60 S subunit were manifested only in the

presence of the complementary small ribosomalsubunit (Nygard & Nilsson, 1989). We weretherefore interested to see if the structure of 18 SrRNA was different in 80 S ribosomes treated withthe two RIPs. Surprisingly, ribosomes depurinatedin the a-sarcin/ricin-loop in 28 S rRNA displayed

Figure 9. In 5 S rRNA, thereactivity of helix I and C, D andE-loops to single-strand modifi-cation was affected by BTI andricin-treatment of 6 S subunits. Referto Figures 2 and 7 for details.


Figure 10. Bases in the 3' majordomain of 18 S rRNA were pro-tected from modification by ricinand BTI-treatment of 80 S ribo-somes. The 80 S ribosomes (0.7 mM)were treated with BTI (16 mM) orricin (0.33 mM) as described for Fig-ure 2. The ribosomes were incu-bated with CMCT and DMS asdescribed in the legends to Fig-ures 2 and 7, respectively.

conformational changes in 18 S rRNA. Such changeswere seen in the 3' major domain where thechemical accessibility of several nucleotides locatedin the hinge regions between helices 36, 37 and 39was reduced after RIP-treatment of the ribosome(Figures 5 and 10). Additional strong structuraleffects were seen at the base of helices 36 and 34,where nucleotides U1506 and U1531 becameprotected from modification. Both ricin and BTIgave similar effects, although the latter inhibitorcaused protection of additional nucleotides in thisregion (Figures 5 and 10B).

Discussion

Almost all RNA sequences affected by BTI andricin-depurination were localized to phylogeneti-cally conserved regions in the rRNA (Gutell et al.,1994; Raue et al., 1990). Moreover, the affectedsequences were mainly localized to regionssuggested to be directly involved in ribosomalfunction such as subunit-subunit interaction, pep-tidyltransferase activity and binding of elongationfactors and tRNA to the ribosome (for a review, seeLieberman & Dahlberg, 1995; Noller, 1991). The

only affected sequences that have not been linked toa specific ribosomal function are domain I and theupper part of domain V. However, accumulatingdata indicate that domain I is involved in thedynamics of the working ribosome (Brow & Noller,1983; Bullard et al., 1995; Holmberg et al., 1994a;Stade et al., 1995). Thus, the experimental datasuggest that BTI and ricin-treatment of ribosomesaffected the structure of rRNA regions that areimportant for ribosomal function.

Effect of BTI and ricin on functional domains

Some of the nucleotides that were affected by BTIand ricin-treatment were located in regions of therRNAs that have been implicated in elongationfactor binding. The BTI and ricin-dependent re-activity changes seen in helices 43 and 44 in 28 SrRNA coincided with such sequences (Holmberg& Nygard, 1994) as did the reactivity changesobserved in the C and D-loops in 5 S rRNA and inhelices 36 and 39 in 18 S rRNA (Holmberg et al.,1992; Holmberg & Nygard, 1994). In E. coli 16 SrRNA the latter region contains the binding-sitefor spectinomycin (Brink et al., 1994; Johanson &


Hughes, 1995; Makosky & Dahlberg, 1987; Moazed& Noller, 1987b; Sigmund et al., 1984). Spectino-mycin interferes with the interaction of theribosome with EF-G (Anderson et al., 1967) mainlyby prolonging the factor-ribosome interaction phaseof the EF-G cycle (Bilgin et al., 1990). It has beensuggested that the effect is due to a stabilization ofhelix 36 in a base-paired conformation (Brink et al.,1994). Our data show that helix 36 becomes lessaccessible for single-strand-specific modificationafter ricin and BTI-treatment of the ribosomes(Figure 10). However, as ricin-treatment of 80 Sribosomes increases the turnover of the ribo-some·eEF-2·GTP complex (Nilsson & Nygard, 1986;Nygard & Nilsson, 1989), a stabilization of helix 36,as a result of depurination of the a-sarcin/ricin-loopin 28 S rRNA, did not prolong the interaction ofeEF-2 with the ribosome.

Ricin and BTI-treatment of the ribosomal par-ticles caused structural changes in helices 43 and 44.It has been suggested that this is a conformationalswitch region that undergoes discrete changesduring the elongation cycle (Cundliffe, 1986;Egebjerg et al., 1989). Nucleotides in the region areinvolved in elongation factor binding (Holmberg &Nygard, 1994; Moazed et al., 1988; Skold, 1983) andhave also been linked to the ribosomal GTPaseactivating centre (Beauclerk et al., 1985; Egebjerget al., 1989). Both ricin and BTI-treatment selectivelyinhibit the eEF-1 a-dependent hydrolysis of GTP,while leaving the eEF-2 mediated hydrolysisuninhibited (Nilsson et al., 1986; Nilsson & Nygard,1986). These observations are consistent with theidea that the activity of the ribosomal GTPhydrolase is influenced by conformational changesin this region of the rRNA.

Accumulated genetic and biochemical evidenceimplicate a central role for domain V of 23 S/28 SrRNA in peptidyltransferase activity and for main-taining translational accuracy (Douthwaite, 1992;Hall et al., 1988; Lieberman & Dahlberg, 1995;Moazed & Noller, 1987a, 1989; O’Connor &Dahlberg, 1995; Porse & Garrett, 1995; Rodriguez-Fonseca et al., 1995; Steiner et al., 1988). Thestructure of the 28 S rRNA around the central ringof domain V was different in RIP-treated andcontrol subunits. Furthermore, ricin and BTI causedinhibitor-specific reactivity changes in helices 78and 96 (Figure 3B). The central part of helix 78is suggested to be the catalytic centre of thepeptidyltransferase (Leviev et al., 1994; Rodriguez-Fonseca et al., 1995). As both BTI and ricin-treatedribosomes are capable of catalysing peptide bondformation (Asano et al., 1986; Montanaro et al., 1973;Nilsson et al., 1986; Nilsson & Nygard, 1986),neither the general nor the RIP-specific structuralchanges seen in helix 78 and in the rest of thepeptidyltransferase domain seem to interfere withpeptide bond formation. However, it is possible thatthese structural alterations interfere with the rate ofpeptide bond formation.

The peptidyltransferase activity is affected byribosomal components outside of domain V. Such

an effect is seen by the interaction between thesmall and large subunits (Monro, 1967; Porse &Garrett, 1995). We have previously observed thatassociation of the 40 S and 60 S subunits alters thechemical modification pattern of 28 S rRNA inregions that have been functionally and/or struc-turally linked to the peptidyltransferase domain(Holmberg et al., 1994a). This supports the idea thatthe peptidyltransferase centre acquires its correctstructure after formation of the complete ribosome.Interestingly, BTI and ricin-dependent changes inthe modification patterns were also found in theseregions of 28 S rRNA. It is possible that theseRIP-dependent changes could interfere with theinteraction of the two subunits during theelongation cycle and therefore interfere with thespeed of peptide bond formation.

5 S rRNA also stimulates peptidyltransferaseactivity (Dohme & Nierhaus, 1976). 5 S rRNA istopographically linked to the peptidyltransferasecentre via a juxtaposition of the D-loop in 5 S rRNAand the apical loop of helix 94 in domain V(Dontsova et al., 1994). As ricin and BTI-treatmentaffected the chemical accessibility of both theD-loop and helix 94, these two structural elementsseemed to be more loosely integrated in RIP-treated60 S subunits and ribosomes (Holmberg et al., 1992).

Two regions in domain II that are structurallylinked to the central ring of domain V (Douthwaiteet al., 1985, 1989; Mitchell et al., 1990; Moazed &Noller, 1987a) were also affected by BTI andricin-treatment of subunits. Interestingly, the RIPsaffected the chemical accessibility of the rRNA inthese two regions in opposite ways (Figure 3A).Thus, the apical loop of helix 31 was specificallyexposed in the BTI-treated 60 S particles but becameless accessible for chemical modification in thericin-treated subunits. Similarly, the reactivity ofhelices 45 and 46 was differently affected but hereBTI-treatment resulted in protections while ricin ledto exposure of nucleotides. These two regions couldtherefore be structural determinants of the BTI andricin-treated subunits. The accumulated data showthat maintenance of a coordinate structural arrange-ment of the peptidyltransferase and its associatedregions is important for ribosomal function in theelongation cycle.

Implication for ribosomal function

During the elongation cycle the ribosome recipro-cates between a pre and post-translocation confor-mation. The ability to form the pre andpost-translocation type of particle is intrinsic to theribosome and both types of ribosomes can beformed in the absence of tRNA and mRNA (Nygard& Nilsson, 1989). Thus, the ribosome itself is adynamic and flexible particle in which thestructural changes are required for ribosomalfunction. Prokaryotic ribosomes are present in twodifferent forms, a contracted and an extended form(Hapke & Noll, 1976). The difference resides in thelarge ribosomal subunit and seems to be dependent


on different conformations of the 23 S rRNA (Burmaet al., 1984; van Diggelen & Bosch, 1973; vanDiggelen et al., 1973). The interconversion betweenthe two forms of 23 S rRNA is influenced by EF-G,(Spirin et al., 1987) and it has been suggested thatthis interconversion is the driving force in thetranslocation step of the elongation cycle (Spirin,1967).

Due to the structural flexibility of the ribosomalparticles, an analysis of the rRNA structure in adynamic population of ribosomal particles willresult in an average structure unless the equilibriumis shifted in favour of one particular conformation.Functional analysis indicates that depurination ofthe a-sarcin/ricin loop by BTI or ricin locks thelarge ribosomal subunit in a particular state bydestroying its dynamic flexibility. This preventsribosomes containing depurinated large ribosomalsubunits from further progression through theelongation cycle (for a review, see Nygard &Nilsson, 1990). The structural alterations seen in therRNAs after BTI or ricin-treatment (exposures,protections and new natural stops) are alsoconsistent with the idea that the ribosome is lockedin a specific conformation. The rRNA containsseveral so-called ‘‘conformational switch’’ regions(helices 43 and 44 in 28 S rRNA, helix 36 in 18 SrRNA and 5 S rRNA: Johanson & Hughes, 1995;Rosendahl & Douthwaite, 1993; Wu & Marshall,1990 and references therein). There was a strikingcorrelation of the inhibitor-related changes in themodification pattern and the location of these‘‘switch regions’’. This suggests that the depurina-tion of A4256 affected the dynamic properties ofrRNA regions essential for the ability of theribosome to reciprocate between the pre andpost-translocation states. The chemical modificationdata presented here indicate that BTI and ricin-treated ribosomal particles have partly differentstructures. Similarly, treatment of ribosomes witha-sarcin, a nuclease that specifically cleaves thephosphate backbone of 28 S rRNA adjacent toA4256, results in a ribosomal protein topographythat is different from that in ricin-treated ribosomes(Terao et al., 1988).

How can depurination of the same base bydifferent RIPs lock the ribosome in discreteconformations? The solution of the three-dimen-sional structure of a 29 nucleotide RNA fragmentidentical with the a-sarcin/ricin region (Szewczaket al., 1993) has shed light on the position of theidentity elements required for recognition bya-sarcin and ricin. The fragment has a compactstructure with a lot of unusual base-pairs andbase-stacking interactions, leaving A4256 and G4257unpaired in a tetraloop. While depurination by ricinis dependent only on the structure of the tetraloopitself other features in the region i.e. a bulged G inthe helix (G4251 in mouse 28 S rRNA) are essentialfor the catalytic action of a-sarcin (Szewczak et al.,1993 and references therein). As suggested by Woolet al. (1992), a-sarcin could induce in the rRNA thestructure required for recognition.

BTI and a-sarcin have similar effects on ribosomalfunction (Fernandez-Puentes & Vazquez, 1977;Nilsson et al., 1986). Moreover, both inhibitors havetheir maximum catalytic activity at low concen-trations of Mg2+ (Figure 1 and see Brigotti et al.,1989; Endo & Wool, 1982), while ricin-depurinationof the a-sarcin/ricin loop was independent of theMg2+ concentration (Figure 1 and see Brigotti et al.,1989). Thus, it is possible that BTI, like a-sarcin,requires other structural elements located outsidethe a-sarcin/ricin loop for recognition. If so, BTI, inanalogy with a-sarcin, would recognize the loopafter melting of the helix. The increased catalyticcapacity at low concentrations of Mg2+ could thenresult from a decreased stability of the helix atlower concentrations of Mg2+. According to thismodel the observed structural changes seen in therRNA would be induced by the ribosome inactivat-ing protein(s). An alternative explanation would bethat, due to the inherent dynamic properties of theribosomal particles, a population of unprogrammedribosomal particles describes an equilibrium be-tween different conformations. The equilibriumcould be shifted in favour of one or the otherconformation as seen in the case of elongation factorbinding (for a review, see Nygard & Nilsson, 1990).According to this model BTI and ricin wouldprimarily interact with ribosomes in differentconformational states (Nygard & Nilsson, 1990). Thesubsequent depurination leads to a shift in theequilibrium so that the majority of the ribosomesbecome trapped in a defined conformation. Thehigher activity of BTI at a low concentration of Mg2+

would in this case result from a shift in the basicequilibrium of the ribosomal population at thereduced concentration of Mg2+. However, if this wasthe only cause of the structural differences seen inthe rRNA, the chemical modification patternsobtained with each RIP would be similar irrespec-tive of the Mg2+ concentration. As this was not thecase, secondary structural changes must be inducedby the RIPs. In this case the structural flexibility ofthe rRNA and thereby the extent of RIP-inducedsecondary effects would be set by the influence ofMg2+ on the rigidity/flexibility of the rRNA.

It is important to remember that, irrespective ofthe mechanisms responsible for the observedstructural differences, ribosomes containing BTIand ricin-treated large ribosomal subunits arefunctionally active in many of the partial reactionsinvolved in a normal elongation cycle. The stabilizedconformations found in BTI and ricin-treatedribosomes must therefore represent physiologicallyrelevant conformations that are natural parts of thefunctional ribosome cycle.

Materials and Methods

Chemicals

DMS and CMCT were from Aldrich Chemie(Germany). Deoxy and dideoxy nucleotides were fromBoehringer Mannheim (Germany). [g-32P]ATP and phage


T4 polynucleotide kinase were from Amersham Inter-national (UK). Superscript reverse transcriptase was fromLife Technologies, Inc. (USA). DNA primers, complemen-tary to mouse rRNA sequences in 5 S, 5.8 S, 28 S and 18 SrRNA, were synthesized as described and the specificsequences used for primer annealing were as described(Holmberg et al., 1992, 1994b).

Preparation of translation inhibitors

Ricin was prepared according to Olsnes & Pihl (1973)and the barley translational inhibitor II (BTI) preparedaccording to Asano et al. (1984) was a kind gift from DrBirte Svensson, Carlsberg Laboratory, Denmark.

Preparation of ribosomal particles

Ribosomes were prepared from mouse Ehrlich ascitescells using the method described by Sundkvist & Staehlin(1975). The 60 S and 40 S subunits were separated andisolated according to Nygard & Nika (1982). Complete80 S ribosomes were formed by incubating 40 pmol of60 S subunits and 20 pmol of 40 S subunits in 12.5 ml ofbuffer A (170 mM KCl, 50 mM Hepes-KOH (pH 7.7),1 mM DTT, 0.1 M sucrose) supplemented with 5 mMMgCl2 for five minutes at 37°C (Holmberg et al., 1994a).

Depurination of A4256 in 28 S rRNA with ricin andBTI inhibitor

The Mg2+-dependence of BTI and ricin-depurination ofA4256 was analysed by diluting 20 pmol of 60 S subunitsin 50 ml of buffer A supplemented with 2, 5, 7 or 10 mMMgCl2. BTI or ricin were added to the samples asindicated. After incubation of the samples for ten minutesat 37°C the rRNA was extracted with phenol (Brawermanet al., 1972). The efficiency of depurination at A4256was monitored by primer extension using the primercomplementary to nucleotides G4312 to U4326 in 28 SrRNA.

For the chemical modification experiments, isolated60 S subunits (40 pmol) or reassociated 80 S (20 pmol)ribosomes were used. The ribosomal particles werediluted in buffer A to a final volume of 50 ml and theMgCl2 concentration was adjusted to either 2 mM or7 mM. BTI and ricin were added to the samples at finalconcentrations of 16 mM and 0.33 mM, respectively, andthe ribosomal RNA was depurinated by incubation forfive minutes at 37°C. Alternatively, the ribosomes wereincubated with ricin for five minutes before addition ofBTI.

Chemical modification of rRNA

Chemical modification of the ribosomal RNA wasperformed as described (Holmberg et al., 1994b) using thesingle-strand specific reagents DMS and CMCT. Thereagents were added to the samples in buffer Asupplemented with either 2 mM or 7 mM MgCl2. Thefinal concentration of DMS was 20 or 90 mM and forCMCT the final concentration was 20 or 100 mM. Thesamples, volume 112 ml, were incubated for 15 minutes at37°C, in the case of CMCT, and for five minutes in thecase of DMS. Control samples, incubated in the absenceof chemical reagent but otherwise treated identically, wererun in parallel. The samples were precipitated by ethanol,collected by centrifugation and the RNA was separatedfrom proteins by extraction with phenol (Brawerman

et al., 1972). The isolated rRNA was precipitated withethanol, dissolved in water and stored in small aliquots at− 80°C.

Identification of modification sites

End-labelling of cDNA primers and primer extensionwere performed as described (Holmberg et al., 1992). Theprimer extension products were separated by polyacryl-amide gel electrophoresis and detected by autoradiog-raphy. The autoradiograms were quantified using acomputer-assisted image analysis system (Holmberget al., 1992).

AcknowledgementsWe are indebted to Dr Birte Svensson for supplying

the purified barley translational inhibitor II and to MrsBirgit Lundberg for technical assistance. This work wassupported by the Swedish Natural Science ResearchCouncil, grant B-Bu 8463-307.

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Edited by D. E. Draper

(Received 9 October 1995; received in revised form 6 March 1996; accepted 12 March 1996)

Depurination of A4256 in 28 S rRNA by the Ribosome-inactivating Proteins from Barley and Ricin...

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