interference and hydroxyl radical footprinting using

The EMBO Journal vol.15 no.6 pp.1421-1433, 1996

Defining the enzyme binding domain of aribonuclease Ill processing signal. Ethylationinterference and hydroxyl radical footprinting usingcatalytically inactive RNase Ill mutants

Honglin Li1 and Allen W.Nicholson2Department of Biological Sciences, Wayne State University. Detroit,MI 48202, USA

'Present address: Cardiovascular Research Center, MassachussettsGeneral Hospital and Harvard Medical School, Boston, MA, USA

2Corresponding author

Ethylation interference and hydroxyl radical foot-printing were used to identify substrate ribose-phos-phate backbone sites that interact with the Escherichiacoli RNA processing enzyme, ribonuclease III. TwoRNase III mutants were employed, which bind sub-strate in vitro similarly as wild-type enzyme, but lackdetectable phosphodiesterase activity. Specifically,altering glutamic acid at position 117 to lysine oralanine uncouples substrate binding from cleavage.The two substrates examined are based on the bacterio-phage T7 R1.1 RNase III processing signal. One sub-strate, R1.1 RNA, undergoes accurate single cleavageat the canonical site, while a close variant, Rl.l[WC-L] RNA, undergoes coordinate double cleavage. Theinterference and footprinting patterns for each sub-strate (i) overlap, (ii) exhibit symmetry and (iii) extendapproximately one helical turn in each direction fromthe RNase III cleavage sites. Divalent metal ions (Mg2+,Ca2+) significantly enhance substrate binding, andconfer stronger protection from hydroxyl radicals, butdo not significantly affect the interference pattern. Thefootprinting and interference patterns indicate that (i)RNase III contacts the sugar-phosphate backbone; (ii)the RNase Ill-substrate interaction spans two turns ofthe A-form helix; and (iii) divalent metal ion does notplay an essential role in binding specificity. Theseresults rationalize the conserved two-turn helix motifseen in most RNase III processing signals, and which isnecessary for optimal processing reactivity. In addition,the specific differences in the footprint and interferencepatterns of the two substrates suggest why RNase IIIcatalyzes the coordinate double cleavage of Rl.l[WC-LI RNA, and dsRNA in general, while catalyzing onlysingle cleavage of R1.1 RNA and related substratesin which the scissile bond is within an asymmetricinternal loop.Keywords: dsRNA/dsRBM/ethylation interference/hydroxylradical footprinting/ribonuclease III

IntroductionThe recognition and metabolism of double-stranded RNAare essential events in a wide range of cellular regulatorymechanisms (Nicholson, 1995). The hydrolytic cleavageof dsRNA is catalyzed by a variety of enzymatic activities,

only few of which have been characterized. A well-known,dsRNA-specific nuclease is ribonuclease III of Escherichiacoli [EC 3.1.24]. RNase III participates in ribosomal RNAmaturation and cleaves other cellular transcripts whoseencoded functions participate in the synthesis, translationand turnover of mRNA (Dunn, 1982; Robertson, 1982;Court, 1993; Nicholson, 1995). RNase III also processesmany phage and plasmid transcripts, as well as antisenseRNA-target RNA duplexes (Court, 1993; Wagner andSimons, 1994; Nicholson, 1995). RNase III-like enzymesare ubiquitous in bacteria (Court, 1993; Mitra andBechhofer, 1994; Zuber et al., 1994) and perhaps also ineukaryotes (Meegan and Marcus, 1989; lino et al., 1991;Nicholson, 1995).The broad occurrence of RNase III-like activities and

their involvement in dsRNA function and metabolismhave spurred studies on the mechanism by which RNaseIII recognizes and cleaves dsRNA. RNase III is active asa homodimer, cleaving both strands of dsRNA in acoordinate (but not necessarily concerted) manner, creatingproducts carrying 5'-phosphate, 3'-hydroxyl termini andexhibiting two nucleotide 3' overhangs (Dunn, 1982;Robertson, 1982; Court, 1993; Nicholson, 1995). Thephosphodiesterase activity requires only Mg2+ as a co-factor [Mn2+, Co2+ and Ni2' are allowable substitutes (Liet al., 1993); H.Li and A.W.Nicholson, unpublished]and at least one carboxylate group may be an essentialparticipant (see below). Substrate recognition is dependenton a dsRNA binding motif (dsRBM) (St Johnston et al.,1992) present in the C-terminal portion of the RNase IIIpolypeptide (Court, 1993). Thus, two dsRBMs occur inthe holoenzyme and probably also two catalytic sites.However, precise descriptions of the mechanisms of sub-strate recognition and phosphodiester cleavage are not yetavailable.The specific features in RNase III processing substrates

that confer cleavage reactivity and selectivity are undercurrent study. A common structure is a dsRNA elementof approximately two helical turns. The homodimericnature of the enzyme and the dyad symmetry of dsRNAtherefore suggest a 2-fold symmetric enzyme-substratecomplex. However, non-canonical structural elements andspecific sequences are essential in establishing the precisereactivity patterns exhibited by the diverse collection ofprocessing substrates (Court, 1993; Nicholson, 1995).Identifying the RNA elements that directly interact withRNase III would define the role of sequence and non-canonical secondary structural elements in processingreactivity; explain the relationship between substratelength and processing reactivity; and assess the involve-ment of metal ion in substrate recognition.We present below a phosphate ethylation interference

and hydroxyl radical footprinting analysis of the.RNase IIIinteraction with two substrates derived from the

© Oxford University Press 1421

H.Li and A.W.Nicholson

bacteriophage T7 RI. 1 RNase III processing signal. Onesubstrate (RI.1 RNA) contains an asymmetric internalloop and undergoes single cleavage, while the othersubstrate (R1.1 [WC-L] RNA) is fully base-paired andundergoes coordinate double cleavage. The experimentsemploy two RNase III mutants, which bind substrate in asimilar manner as wild-type enzyme, but are incapableof catalyzing phosphodiester hydrolysis. The ethylationinterference and hydroxyl radical footprinting results pro-vide a preliminary model of how RNase III interacts withspecific processing substrates. This study also shows thatdivalent metal ions (Mg2+ and Ca2+) promote substratebinding, but apparently do not confer binding specificity.

ResultsPurification and properties of the rnc[E117Kl andrnc[E117AI RNase 111 mutantsAn RNase III mutant which can bind substrate but cannotcatalyze cleavage would be useful in mapping enzymecontact sites on substrate and assessing the role of divalentmetal ion in recognition. A previous report described theisolation of the rnc70 mutation, which abolishes processingin vivo (Inada et al., 1989). It was subsequently determinedthat the rnc70 mutation changes glutamic acid to lysineat position 117 in the RNase III polypeptide and evidencewas cited that the mutant protein could bind substrate(Court, 1993). To investigate further the functional con-sequences of amino acid substitution at position 117,recombinant plasmids were prepared which directed thehigh-level expression of the rnc[E117K] or the relatedrnc[EI 17A] RNase III mutant. The proteins wereexpressed in RNase 111- cells to avoid interference fromendogenous enzyme and purified according to a previouslydescribed protocol (Li et al., 1993). Both proteins exhibitedan affinity for poly(I)-poly(C)-agarose comparable withthat of wild-type RNase III and the gel filtration behaviorof both mutants was consistent with a dimeric structure(data not shown).The reactivities of the mnc[E117K] and rmc[E117A]

mutants toward homobifunctional, amine-specific cross-linking reagents also support the dimeric nature of themutant proteins. Mutant or wild-type protein was incubatedwith disuccinimidyl suberate (DSS) (Montesano et al.,1982), the products fractionated by SDS-PAGE and visual-ized by Coomassie staining. In all cases a DSS-dependentspecies is produced which migrates in a position commen-surate with a cross-linked dimer (-52 kDa molecularmass) (Figure 1). A consistently stronger cross-linkingreaction was observed with the rnc[E1 17K] mutant, whichmay be due to the direct participation of the additionallysine residue in the cross-linking reaction. Experimentsinvolving an 10-fold lower protein concentration anddisuccinimidyl tartrate (DST) (Smith et al., 1978), whichhas a shorter cross-linking arm, provided similar results(data not shown, but see March and Gonzalez, 1990).Under these conditions DST specifically detected proteindimers, as its reaction with purified monomeric T7 RNApolymerase did not produce a cross-linked species (datanot shown).

Changing Glu117 to lysine or alanine abolishesRNase 11 processing in vitroThe RNA cleaving activities of the purified mnc[EI17K]and rnc[E 117A] proteins were examined using two sub-

Fig. 1. Cross-linking of the rnc[EI 17K] and rnc[El 17A] RNase IIImutants using DSS. Cross-linking reactions (10 gl volumes) includedI ,ug purified protein (4 luM monomer) and were performed at roomtemperature for 15 min in 10 mM MOPS (pH 7.5), containing250 mM NaCl, 0.1 mM EDTA and 0.1 mM DTT. Reactions werequenched by boiling in SDS gel loading buffer supplemented with0.5 M Tris base, then analyzed by electrophoresis in a 12%polyacrylamide-SDS gel, using a discontinuous buffer system (Studier,1973). Proteins were visualized by Coomassie Blue staining: lanes2-4, wild-type RNase III; lanes 5-7, rnc[EI17A] mutant; lanes 8-10,rnc[E117K] mutant; lanes 2, 5 and 8, protein incubated without DSS;lanes 3, 6 and 9, protein incubated with I ,ug of DSS; lanes 4, 7 and10, protein incubated with 10 jg DSS; lane 1, prestained protein sizemarkers, with the molecular masses (in kDa) indicated.

C AG A

30-C GU AU GA UC GU G-40G CG CA UA U

20- C UU AC UA G

AA U-soA UC G

A AG CG C

io-G UA UG CA U

StpppGGGAGU UOH

R1.1 RNA

C AG AC G

30-U AU GA UC G-40U GG CG CA UA UA U

20-U AA UC GU A-soA UA UC G

A AG CG C

lo-G UA UG CA U-6o

5'pPPGGGAGU UOH

R1 .1 [WC-L] RNA

Fig. 2. Sequence and secondary structure of Ri. 1 RNA and Ri .1 [WC-L] RNA. Ri .1 [WC-L] RNA differs from Ri. 1 RNA by replacement ofthe internal loop with a fully WC base-paired segment (Chelladuraiet al., 1993). The RNase III cleavage sites are shown in Figure 7.

strates based on the T7 phage RI. 1 RNase III processingsignal (Dunn and Studier, 1983). RI.1 RNA (Figure 2A)exhibits an irregular hairpin structure (Schweisguth et al.,1994) and is cleaved at a single site within the asymmetricinternal loop (Nicholson et al., 1988; Chelladurai et al.,1991). Ri.l[WC-L] RNA (Figure 2B) exhibits a fullyWatson-Crick (WC) base-paired internal loop andundergoes coordinate double cleavage (Chelladurai et al.,1993). Under conditions where wild-type RNase III carries

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RNase III-processing substrate interaction

;.-. .

Fig. 3. Processing activities of the rnc[E I 17K] and mnc[E I 17A] proteins. Reactions (10 ,l) included 5'-32P-labeled Rl.l[WC-L] RNA (5000 d.p.m.,0.75 fmol) and the reaction products were analyzed by electrophoresis (23 V/cm) in a 15% polyacrylamide-7 M urea gel followed byautoradiography and radioanalytic imaging. (A) Cleavage assay. Lanes 1-6, wild-type RNase III [rnc(wt)]; lanes 7-12, rnc[E117A] protein; lanes13-18, rnc[E1 17K] protein. Specific conditions were: lanes 1-3, 20 nM rnc(wt) protein (0, 2, 20 min); lanes 4-6, 200 nM rnc(wt) protein (sametimes); lanes 7-9, 60 nM mnc[E1I7A] protein (same times); lanes 10-12, 600 nM rnc[E117A] protein (same times); lanes 13-15, 80 nM rnc[E117K]protein (same times); lanes 16-18, 800 nM rnc[E1 17K] protein (same times). (B) Mutant protein inhibition of wild-type RNase III processing.Increasing amounts of mutant protein were added to reactions containing wild-type RNase III (10 nm) and 5'-32P-labeled R1.1 RNA (5000 d.p.m.,0.75 fmol), followed by incubation for 5 min. Lanes 1 and 9, no enzyme; lanes 2-8, rnc[E I17A] protein (0, 1, 10, 50, 200, 500 and 1000 nM finalconcentrations, respectively); lanes 10-16, rnc[E1 17K] protein (same amounts). The 5' end-containing product of RNase III cleavage is indicated by' 5',' and the position of the xylene cyanol dye marker is indicated by 'XC'.

out efficient processing of 5'-32P-labeled Ri.1 [WC-L]RNA (Figure 3A, lanes 1-6), there is no detectablecleavage by either mutant protein (Figure 3A, lanes 7-12,and 13-18), even with excess protein and prolongedreaction times. Also, neither mutant protein could cleaveRl.1 RNA (data not shown). It is possible that the lackof processing activity reflects a shifted salt optima oraltered divalent metal ion specificity. However, no detect-able processing occurred when (i) the salt concentrationwas lowered; (ii) the Mg2+ concentration was increased;(iii) Mg2+ was replaced by Mn2+; or (iv) a non-ionicdetergent was included (data not shown).To determine whether the rnc[El 17K] and rnc[E1 17A]

proteins can inhibit Ri.1 RNA processing by wild-typeenzyme, mutant protein was combined in reaction bufferwith wild-type enzyme in specific ratios. Substrate wasthen added and the reaction initiated by adding Mg2 .Increasing the mutant:wild-type protein ratio progressivelyinhibits Rl.1 RNA processing, such that 50% inhibitionoccurs at ~10 nM rnc[E117K] protein and at a slightlylower concentration of rnc[E117A] protein (Figure 3B,lanes 4 and 12). A concentration of 10 nM correspondsto a rnc[E1 17K] mutant/wild-type protein ratio of 1:1 and

suggests a mutant protein affinity for substrate comparablewith that of wild-type RNase III under optimal processingconditions. (It is possible that subunit exchange couldoccur under these conditions, which could provide anextra source of inhibition if the heterodimeric species isinactive.)

Substrate binding affinity of the rnc[E117Kl andrnclE117Al RNase 111 mutants, and divalent metalion dependenceThe ability of the mnc[EI17K] and rnc[EI17A] proteinsto bind processing substrate is suggested by their affinityfor poly(I)-poly(C)-agarose and their ability to inhibitwild-type RNase III processing. Gel mobility shift assayswere used to assess directly the substrate binding affinitiesof the mutant proteins. A previous gel shift analysisdemonstrated the formation of a 1:1 complex of wild-typeRNase III and RI. 1 [WC-L] RNA in buffer lacking Mg2+(Chelladurai et al., 1993). To compare directly the mutantand wild-type enzymes, gel shift experiments were per-formed in the absence of Mg2. The results show that themnc[El 17K] (Figure 4B) and rnc[E1 17A] (Figure 4C)proteins both bind RI. 1 [WC-L] RNA, and with a higher

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Fig. 4. Substrate binding by the rnc[E1 17K] and mnc[El 17A] proteinsin the absence of Mg2+. RNase III mutant protein (amounts givenbelow) was incubated with 5'-32P-labeled RI.1 [WC-L] RNA (2x 104d.p.m., 0.3 fmol) in binding buffer lacking Mg2, then electrophoresedin a non-denaturing polyacrylamide gel (see Materials and methods).(A) Wild-type RNase III; (B) rnc[E1 17K] mutant; (C) rnc[E1 17A]mutant. The amount of added protein is indicated at the top of (A).I ng corresponds to 10 fmol dimer. The positions of the xylene cyanol(XC) and bromphenol blue (BP) dye markers are indicated. 'F' and'B' refer to free and enzyme-bound RNA, respectively. The data inthese experiments were included in calculating Kd values (see Table I).

affinity than wild-type RNase III (Figure 4A). With anincreasing amount of either mutant protein there occursessentially complete conversion of free substrate to itsprotein-bound form. In contrast, a substantial level of freeRNA remains even at the highest concentration of wild-type RNase III, with the accompanying radioactive smearpresumably representing dissociation of the enzyme-sub-strate complex during electrophoresis (see also Chelladuraiet al., 1993). Competition gel shift assays, using 32p-labeled Ri.1 RNA or R 1.l[WC-L] RNA and includingincreasing amounts of the other substrate in non-radio-active form, suggest that the two mutant proteins exhibitthe same differential affinities as wild-type RNase III forRl.l RNA and Rl.I[WC-L] RNA (data not shown). Todetermine the dissociation constants (Kd values), the gelshift experiments were performed several times and thedata (including that in Figure 4) quantitated by radio-analytic imaging. The averaged Kd values with standarderrors are provided in Table I. The Kd values of RI. 1 [WC-L] RNA bound to the rnc[E1 17K] and rnc[EI 17A] proteins(4.7 and 4.0 nM, respectively) in Mg2+-free buffer are~6- to 7-fold lower than the Kd value for wild-type enzyme

(29 nM). The reason for the tighter binding of the mutantproteins in the absence of divalent metal ion comparedwith wild-type enzyme is not clear. However, since thechemically different alanine and lysine residues confercomparable binding, the difference must reflect the lossof the carboxyl group, rather than the specific chemicalnature of the substituted residue.The catalytic inactivity of the rnc[E1 17K] and

rnc[Ei 17A] proteins was exploited to determine whetherdivalent metal ion participates in substrate binding. It waspreviously shown that in the absence of Mg2+ wild-typeRNase III cannot engage RI. 1 RNA in a stable gel-shifted complex (Chelladurai et al., 1993). The gel shiftexperiments displayed in Figure 5 indicate that neithermutant protein can engage RI. 1 RNA in an electro-phoretically stable complex in Mg2'-free buffer, sincewith increasing protein concentrations there is no changein the amount of free RNA (Figure SB and D). However,including 5 mM Mg2+ in the binding and electrophoresisbuffers affords stable binding of RI.1 RNA to both mutantproteins (Figure 5A and C). A Mg2+ concentration of5 mM represents a near-saturating value with respect tobinding enhancement, since raising the concentration to10 mM provides essentially the same result (data notshown). Table I provides the Kd values (5 mM Mg2+) forR1.1 RNA binding to the rnc[E117K] and rnc[E117A]proteins, where it is seen that the two mutant proteinsexhibit comparable affinities for RI. 1 RNA. The effect ofMg2+ on mutant protein binding to Ri.1[WC-L] RNAwas also determined. In this case the binding enhancementcould be directly measured, since a gel-shifted complexoccurs in the absence of Mg2+ (see Figure 4). Themeasured Kd values (Table I) indicate that Mg2+ enhancesbinding of RI. 1 [WC-L] RNA to either mutant protein by3- to 4-fold.Can divalent metal ions other than Mg2+ enhance

substrate binding? Gel shift assays which substituted Ca2+for Mg2+ provide a single shifted RNA-protein complexwhich exhibits a mobility similar to that of the Mg-2+stabilized complex (data not shown). Since Ca2+ is inactiveas a catalytic cofactor (Li et al., 1993), it could be usedto determine the effect of divalent metal ion on wild-typeRNase III binding to substrate. The Kd values (5 mMCa2+) given in Table I show that Ca>2 is comparable withMg2+ in enhancing RNA binding to wild-type as well asmutant protein. Essentially the same values were obtainedat 10 mM Ca2+ (data not shown). In summary, both Mg2+and Ca2+ confer comparable stabilities with the complexof mutant or wild-type enzyme with RL.I RNA orRi.1[WC-L] RNA. Divalent metal ion therefore has agreater stabilizing effect on Rl.1 RNA binding than onRi.1[WC-L] RNA binding. Gel shift experiments usingMn2+ or Zn2+ were inconclusive, as metal precipitatesformed during electrophoresis (data not shown).The gel mobility shift assays also reveal a specific

influence of divalent metal ion on RNA electrophoreticmobility. Whereas in the absence of divalent metal ionRI.1 RNA migrates as a single band, the addition ofMg2+ produces a closely-spaced doublet (compare Figure5A and B, and C and D). A less distinct, more closelyspaced doublet is seen with Ri.1[WC-L] RNA in thepresence of Mg2+ and Ca2+ (data not shown). However,the doublet is not observed when electrophoresis is per-

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Table I. Binding affinities of Ri. 1 RNA and R 1.1 [WC-L] RNA for wild-type and mutant RNase III. in the presence and absence of divalent metalion'

Protein Rl.l RNA R 1. [WC-L] RNA

-Mg-+ +Mg2+ +Ca2+ -Mg-2 +Mg2 +Ca+

Wild-type - - 1.8 + 0.3 29 6 - 1.8 + 0.2rnc[E117K] - 2.3 + 0.2 1.6 + 0.2 4.7 + 1.7 1.6 + 0.1 1.8 + 0.3rnc[E117A] - 1.6 + 0.3 1.7 + 0.3 4.0 + 0.5 1.0 1 0.2 1.6 + 0.2

aThe binding of 5'-32P-labeled RNA to wild-type or mutant RNase III was quantitated by radioanalytic imaging of gel shift assays (electrophoresisperformed at 5-6°C) as described in Materials and methods. Specifically, the amount of free RNA was measured as a function of proteinconcentration and used to calculate the fraction of RNA bound (v). The slope of the lines in double-reciprocal plots (Carey et al., 1983) provided theKd values. The listed Kd values (nM) represent the average of two or more independent determinations and the maximum error is given. Where novalue is reported either means binding was too weak to be measured by this assay or that substrate processing by wild-type enzyme (in the presenceof Mg2+) prevented measurement.bThe Kd value for R1.1[WC-L] RNA binding to wild-type RNase III in the absence of Mg2+ agrees, within experimental error, to the previouslydetermined value of 35 nM (Chelladurai et al., 1993).

&~'~'A 45~5~A ~ *It~ess~sIW, aws -p

Fig. 5. Mg2+ stabilization of the RNase 111-substrate complex.5'-32P-labeled Rl.l RNA (2X104 d.p.m., 0.3 fmol) was combinedwith rnc[El 17K] protein (amounts given at the top of panel A) andnon-denaturing gel electrophoresis performed (see Materials andmethods). (A) mnc[E I17K] protein (5 mM Mg2); (B) rnc[E1 17K]protein (no Mg2+); (C) rnc[EI 17A] protein (5 mM Mg2+); (D)rnc[E l 17A] protein (no Mg2+). 'F' and 'B' refer to the unbound andbound RNA fraction, respectively, and the position of the xylenecyanol (XC) dye is indicated.

formed at room temperature (data not shown). Perhapsdivalent metal ion stabilizes two conformations of R1.1RNA, which exhibit different gel mobilities, but whichundergo fast exchange in the absence of Mg2+ or Ca2+.In this regard, an NMR analysis has revealed that Ri.1RNA has one or more specific Mg2+ binding sites associ-ated with the internal loop and perhaps also the tetraloop(Schweisguth et al., 1994).

Identification of substrate phosphodiester linkagesimportant for RNase 111 bindingEthylation interference analysis can identify RNA phos-phodiester linkages whose covalent modification inhibits

protein binding (e.g. see Gott et al., 1993). The identifiedlinkages (hereafter called 'pro-interfering' phospho-diesters) are inferred to participate in functionally import-ant protein-RNA contacts. 5'-32P-labeled Ri.1[WC-L]RNA was treated with ethylnitrosourea (ENU) such that20% modification was achieved (see Materials andmethods) then combined with wild-type RNase III (withoutMg2+) or with the rnc[E1i7A] or rnc[E117K] protein(with or without Mg2+). The protein-bound and free RNAswere separated by non-denaturing gel electrophoresis andthe purified RNAs incubated in mild alkali to cleavephosphotriester linkages. The products were fractionatedby sequencing gel electrophoresis and analyzed by auto-radiography. Figure 6A displays the autoradiographicpattern of ethylated RI. 1 [WC-L] RNA, bound in theabsence of Mg>2 to either wild-type, rnc[EI17A] orrnc[EI17K] protein. Figure 6B displays the interferencepatterns for ethylated Ri.1[WC-L] RNA bound in thepresence of Mg2+ to rnc[EI17A] or rnc[EI17K] protein.Figure 6C displays an experiment essentially the same asthat in Figure 6B, except that electrophoresis was per-formed for a longer amount of time in order to examine pro-interfering phosphodiesters near the RI. 1 [WC-L] RNA 3'end. Similar experiments were performed using 5 -32p-labeled RI.1 RNA (data not shown).

First, the autoradiographic patterns reveal that all ofthe phosphodiester linkages in Ri.1 [WC-L] RNA aresusceptible to ethylation, indicating solvent accessibilityof the phosphodiester oxygens (lanes 'F', Figure 6).Second, ethylation of specific phosphodiester linkagesinterferes with RNase III binding, as the protein-boundRNA fraction (lanes 'B', Figure 6) lacks (or is under-represented in) specific ENU-dependent cleavage products,compared with those derived from the unbound RNAfraction (lanes 'F', Figure 6). Third, the interferencepatterns (-Mg2+) for wild-type enzyme and the two mutantproteins are essentially the same (Figure 6A). Therefore,the rnc[EI17K] and mnc[EI17A] proteins bindR 1.l[WC-L] RNA in the same or closely similar manneras wild-type RNase III. Fourth, the interference patternsfor the mutant proteins are not significantly changed bythe presence of Mg21 in a qualitative significant manner[compare Figure 6A, El 17A and El 17K (-Mg2+) experi-ments with Figure 6B, (+Mg2+) experiments]. Thus,although Mg2+ enhances Ri.1[WC-L] RNA binding toRNase III, it cannot overcome the inhibitory effect of

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Fig. 6. Ethylation interference analysis of RNase III binding to Rl.l[WC-L] RNA. 5'-32P-labeled Rl.l[WC-L] RNA (106 d.p.m., 0.15 pmol) wastreated with ENU (37°C for 90 min), then purified and combined with either wild-type, rnc[El 17K] or rnc[El 17A] protein in the presence orabsence of Mg2+. Enough protein was added such that -50% of the RNA engaged in a gel-shifted complex. Non-denaturing gel electrophoresis wasperformed, the free and bound RNA fractions purified, briefly incubated at pH 9 and equal amounts of 32P-radioactivity analyzed by sequencing gelelectrophoresis (see Materials and methods). (A) (-Mg2, 12% gel). The first set of eight lanes (experiment 1) displays the interference pattern forwild-type (wt) RNase III; the second set of lanes (experiment 2), the rnc[El 17A] protein pattern; and the third set of lanes (experiment 3), thernc[E1 17K] protein pattern. For each experiment, the first lane displays the RNase U2 (A>G-specific) sequencing ladder; the second lane, analkaline hydrolysis ladder, then (in groups of two lanes each), the free (F), bound (B) and free (F) RNA fractions. The positions of the tetraloopGCAA sequence and the RNase III cleavage sites at positions 20 and 48 are indicated on the left hand side. (B) (+Mg2+; 15% gel). The first set ofeight lanes is wild-type RNase III (-Mg2+,) the second set of lanes, rnc[E1 17A] protein (+Mg2+) and the third set of lanes, rnc[E1 17K] protein(+Mg2+). (C) Repeat of experiment in (B), except that electrophoresis was performed for a longer amount of time, in order to visualize pro-interfering phosphodiesters near the 3' end.

specific phosphodiester ethylation. Essentially the sameresults were obtained with 5 '-32P-labeled RI. 1 RNA, usingmutant protein in the presence of Mg2+ (data not shown).No interference was able to be determined for mutant orwild-type protein in the absence of Mg2, since the Ri. 1RNA-protein complex does not survive non-denaturinggel electrophoresis (Figure 5).

Figure 7 displays the positions of the pro-interferingphosphodiesters in RI. 1 [WC-L] RNA and RI. 1 RNA. ForRi1.[WC-L] RNA, the pro-interfering phosphodiestersexhibit a degree of symmetry about the RNase III cleavagesites. Specifically, there occurs a contiguous run of sixpro-interfering phosphodiesters (ApC14-CpAI9 and CpC43-

ApU48) on the 5' side of each RNase III cleavage site(Figure 7A). Although no pro-interfering phosphodiestersoccur in the GCAA tetraloop, two pro-interfering phospho-diesters (UpC31 and GpU39) occur near this motif and areapproximately one A-helical turn from the two phospho-diesters centered between the RNase III cleavage sites.Three pro-interfering phosphodiesters, CpU57, UpU58,UpC59, occur near the 3' end, also approximately onehelical turn from the RNase III cleavage sites. The UpC59phosphodiester is symmetrically related to the UpC31phosphodiester. The identification of these two groups ofpro-interfering phosphodiesters distal to the processingsites indicates that stable binding of RNase III depends

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A BC A C A

G A G AC G 30iC G

30-U A U AU G U GA U A UC G-4o C GU G U G-4oG Co GCoA U *A U0

_A U 20 C U20 -U A U A

° C G- * A GAUA-*oU A

OC G° C G UA A A AG CGG C*G C G C

lo-G U lo- G UAU A UGC G CA U-6o A U

S'pppGGGAGU UOH s5ppGGGAGU UOH

Fig. 7. Ethylation interference diagrams of Rl1. [WC-L] RNA (A) andRI. 1 RNA (B). The closed and open circles indicate sites of stronglyor weakly pro-interfering phosphodiesters, respectively. Arrowsindicate RNase III cleavage sites. Position assignments took intoaccount the fact that the pH 9 cleavage products of ENU-treated RNAexhibit slightly slower electrophoretic mobilities, compared with thealkaline ladder products, due to ethyl groups at the 3' end of theRNAs (Vlassov et al., 1981). The 5' end-proximal portion (nt 1-10) ofRI. 1 [WC-L] RNA could not be analyzed, due to the loss of shortRNAs dufing ethanol precipitation (see also Vlassov et al., 1981).

on specific phosphodiester groups positioned approxi-mately one helical turn in either direction from thecleavage sites.A similar array of pro-interfering phosphodiesters is

observed in RI. 1 RNA (Figure 7B). However, comparedwith Ri.1[WC-L] RNA, there occurs several additionalpro-interfering phosphodiesters (UpC20-ApG23) whichmap within and above the left side (5') segment of theinternal loop. Apparently, the presence of the asymmetricinternal loop perturbs the RNase III-substrate interactionsuch that ethylation of these additional sites also signific-antly weakens enzyme binding (see Discussion). Theabsence of perfect symmetry in the interference patternsfor either substrate probably reflects the imperfect 2-foldsymmetry-in sequence and in structure-for both RI.1RNA and Rl.l[WC-L] RNA.

Identification of RNase III-protected ribose groupsby hydroxyl radical footprintingHydroxyl radical footprinting can identify ribose residuesthat are protected by bound protein or are sequesteredfrom solvent by RNA tertiary structure (Latham andCech, 1989; Rosendahl and Douthwaite, 1993). Hydroxylradicals are small, neutral and highly reactive, and as aconsequence are relatively insensitive to RNA sequenceand secondary structure. 5'-32P-labeled RI.1 RNA orRl.1[WC-L] RNA was bound to wild-type or mutantRNase III in the absence or presence of Mg2> andthe RNA-protein complexes briefly exposed to hydroxylradicals. The RNA products were electrophoresed in a

sequencing gel and visualized by autoradiography. Foot-prints of wild-type RNase III (without Mg2+) and thernc[EI17A] protein (with and without Mg2+) on Ri.1RNA are shown in Figure 8A, while Figure 8B displaysthe footprint of the rnc[E117K] protein on Ri.1[WC-L]RNA in the presence and absence of Mg.2+

Predominantly uniform cleavage of Rl.1 RNA andRi.1[WC-L] RNA occurs in the absence of protein ('-'lanes in Figure 8A and B), indicating that all riboseresidues are solvent-accessible. In the absence of Mg2+there is no detectable RI. 1 RNA footprint for either wild-type or nc[IEl 17A] protein (experiments 1 and 3, Figure8A). However, under the same conditions weak protectionis observed with RI. 1 [WC-L] RNA (Figure 8B) which isconsistent with its greater affinity for RNase III in theabsence of Mg2+ (Table I). The inclusion of Mg2+ in thernc[EI 17A] and rnc[EI 17K] protein footprinting experi-ments confers significant protection of specific sites inRl.I RNA and Ri.l[WC-L] RNA (compare experiments2 and 3 in Figure 8A and B). However, Mg2+ does notprovide measurable protection in the absence of protein('-' lanes in Figure 8A and B). Thus, the enhancedprotection afforded by divalent metal ion reflects a stabil-ized enzyme-substrate complex. Moreover, the protectiveeffect is not Mg2+-specific: substitution of Ca2+ for Mg2+in the mnc[EI 17A] protein experiment provides essentiallythe same footprint (data not shown). Since Ca2+ is inactiveas a catalytic cofactor (Li et al., 1993), it was also addedto footprinting reactions containing wild-type RNase III.The observed protection patterns are essentially the sameas the patterns for either mutant enzyme in the presenceof Ca2+ or Mg2+ (data not shown). The Ri.1[WC-L]RNA protection patterns in the absence or presence ofMg2+ also indicate that Mg2+ does not qualitatively alterthe protection pattern, but increases the extent of protectionat otherwise weakly protected sites. Thus, these experi-ments suggest that divalent metal ion does not conferenzyme binding specificity of RNase III to RI.1 RNAand RI.I [WC-L] RNA.

Figure 9A and B summarizes the positions of theprotein-protected ribose residues (in the presence of Mg2+)in the Rl.I RNA and Rl.I[WC-L] RNA structures. Thediagrams incorporate the results of additional experiments,which used gels with higher acrylamide concentrationsand shorter electrophoresis times, allowing examinationof protected sites near the RNA 5' ends. The patternsexhibit an imperfect symmetry, which in part reflectsthe imperfect 2-fold symmetry of the two RNAs. TheRI. 1 [WC-L] RNA region most strongly protected byRNase III from hydroxyl radical attack is symmetricallycentered about the processing sites. Specifically, there aretwo segments containing four contiguous, highly protectedresidues (UI7-U20 and U45-U48) which map immediatelyupstream from each RNase III cleavage site (Figure 9B).Downstream (3') of these segments are two protectedsequences, G24-U26 and U52-A54. Since these sequencesare approximately one-half helical turn downstream fromeach processing site, they are situated on the helix faceopposite the processing sites. Significant protection alsooccurs at specific sites up to one helical turn from theprocessing sites [C31, G38 and U39] (Figure 9B). Thepositions of these sites supports the interference experi-

1427


- w~~~~~~~M- - '

Fig. 8. Hydroxyl radical footprinting of the Rl.1 RNA- and the Ri.l[WC-L] RNA-RNase III complex. 5'-32P-labeled Ri.1 RNA was combinedwith wild-type or mutant RNase III, in the presence or absence of Mg2+ and incubated with hydroxyl radical-generating reagents. The products wereanalyzed in a polyacrylamide sequencing gel and autoradiography performed (see Materials and methods). (A) The footprints of wild-type (wt) andrnc[E117A] protein bound to R1. RNA, in the absence or presence of Mg2+. The first set of six lanes is wild-type RNase III without Mg2+; thesecond set of eight lanes is the rnc[E1 17A] protein with Mg> and the third set of eight lanes is rnc[E1 17A] protein without Mg2. (-) and (±)indicate the absence and presence of protein, respectively. The first and second lanes in each set are the RNase U2 (A>G) and alkaline ladders,respectively. The position of the GCAA tetraloop, the RNase III cleavage site at nts 47 and 20 are indicated on the left-hand side. In experiment 1,the stimulated cleavage at U45 was not reproducible. (B) The footprint on RI. 1 [WC-L] RNA of rnc[E1 17K] protein in the absence or presence ofMg2+. The first set of seven lanes is the wild-type RNase III footprint, the second set of 8 lanes is the rnc[EI17K] protein footprint (_Mg2+), andthe third set of eight lanes is the rnc[E1 17K] footprint (+Mg2+). Lanes 1 and 3 in the first set, and lanes 2 in the second and third set are alkalineladders. Lane 2 in the first set, and lane 1 in the second and third set, are RNase U2 reactions. (C) Analysis of the ascorbate/hydrogen peroxide-dependent cleavage products of 5'-32P-labeled Rl .1 [WC-L] RNA. Reactions were carried out using the rnc[E1 17K] protein and applying thehydroxyl radical footprinting protocol, except that Fe(II)-EDTA was omitted. Reactions were supplemented as indicated with 0.1, 1 or 10 mM Mg2+(lanes 5-8) or Ca2+ (lanes 10-13). Lanes 8 and 13 (10 mM divalent metal ion) lacked ascorbate and hydrogen peroxide. Lanes I and 2 are theRNase U2 and alkaline ladders, respectively. The position of the GCAA tetraloop and the RNase III cleavage sites (U20 and U48) are indicated onthe left-hand side.

ments in indicating that RNase III directly contacts sub-strate over two helical turns.The protected sites in RI. 1 RNA (Figure 9A) are largely

similar to that of RI.1 [WC-L] RNA. However, significantdifferences occur in the internal loop region: RI.1 RNAdisplays only five protected sites (A13-A17), occurring onthe left (5') side and upstream of the internal loop, whereasRi1.[WC-L] RNA displays a total of 10 protected sites inthe same region (G11-U20). In addition, residue U47 is not

appreciably protected, whereas the corresponding nucleot-ide in R1. 1 [WC-L] RNA (U48) is. Thus, bound RNase III isless able to protect residues in an internal loop than withthe corresponding dsRNA segment in RI. 1 [WC-L] RNA.Finally, whereas U52-A54 is protected in RI. 1 [WC-L] RNA,the corresponding sequence in RI. 1 RNA is not. The alteredprotection pattern in the internal loop region may reflect thesource for the differing processing reactivity patterns of thetwo RNAs (see also Discussion).

1428

..dvim

Mm',


AC A

G A030-C G

U AU GoA UoC G

OU G-400oG CoOG CA UoA UC

*20-C UoU A-C U.w*A Gi

A* A U-so*A U*C G

*A AG CG C

io-G UoA UG CA U

5'pppGGGAGU UOH

BC A

G AoC G

30-U AU GoA UoC G-4o

oU GeoG CooG CA UA U

*A Ue*20-U AO* U**C G*U A-soQA UQA UOC G

*A AooG CoG C

io-G UA UG CA U-6o

5pppGGGAGU UO.

CC A

G AC G

30-U AOU GeAUC G-4oU GG CG CA U

*A U*A U

020-U A-*A UC GeU A-so.AUAUC G

A AG CG C

io-G UAUG CA U60

5SpppGGGAGU UOH

Fig. 9. Diagrams of Rl.l RNA (A) and Rl.l[WC-L] RNA(B) showing the ribose residues protected by protein from hydroxylradicals. Sites protected from hydroxyl radical attack were assigned byalignment using an alkaline ladder and RNase U2 (A>G) cleavage.Position assignments took into account the fact that hydroxyl radicalattack causes the loss of the damaged nucleotide, creating 5 '-32P-labeled products which terminate with 3'-phosphates. In assessing therelative amount of protection, the tetraloop nucleotides were assumedto exhibit comparable reactivities in the absence or presence ofprotein. The filled and open circles indicate sites of strong and weakprotection, respectively. The stars indicate sites of enhanced cleavagein the presence of protein and Mg2+ (see also Figure 9C). Residue U48is not only protected from hydroxyl radical attack, but is sensitive toascorbate/hydrogen peroxide. This reactivity assignment was possible,since only a fraction of the RNA was modified. (C) Diagram ofRi.1[WC-L] RNA, showing the ascorbate/hydrogen peroxide-sensitivesites. The positions of the nucleotides representing the 3' ends of the5'-32P-labeled cleavage products are indicated by filled circles; the sizeof the circle indicates the relative amount of product. Assignment ofsites avoided any assumptions about the cleavage reaction mechanism,which is unknown (see Results). However, if the hydroxyl radicalchemistry applies, all sites would be shifted one nucleotide towardsthe 3' end. The RNase III cleavage sites are indicated by arrows.

Enhanced RNA cleavage in the RNase III-substratecomplex is independent of Fe(ll) and dependent onascorbate and hydrogen peroxideSites of enhanced cleavage are seen in the hydroxylradical footprints of Ri.1 RNA and Ri.1[WC-L] RNA(experiment 2, Figure 8A and experiment 3, Figure 8B).These sites are indicated by stars in Figure 9A and B.Enhanced cleavage only occurs in the presence of proteinand Mg>2. Perhaps the substrates undergo a conforma-tional change upon binding metalloenzyme, providingselectively enhanced susceptibility to hydroxyl radicalattack from solution. It is also possible that enhancedcleavage occurs by a separate mechanism. Experimentswere performed in which component hydroxyl radicalreagents were added singly or pairwise to the R1.1 RNA-rnc[El 17A] protein-Mg2> complex. The single additionof Fe(II)-EDTA, ascorbate or hydrogen peroxide did notpromote the selective cleavage. Also, no RNA ladder wasproduced in the absence of protein (data not shown). Thelack of a ladder indicates the absence of hydroxyl radicalsin solution-consistent with the Fe(II) independence-aswell as the absence of alkaline-induced cleavage. However,

coaddition of hydrogen peroxide and ascorbate to thecomplex promoted selective cleavage (Figure 8C). Severalof the products correspond to those produced in thehydroxyl radical footprinting reaction (compare Figure 9Bwith C). The cleavage products possess 3'-phosphatetermini, since they comigrate with the alkaline ladderproducts (Figure 8C). Thus, the reaction is not directlyrelated to the RNase III catalytic mechanism (e.g. fromcontaminating wild-type enzyme or activation of thernc[EI 17A] mutant protein). The ascorbate/peroxide-dependent cleavage reaction requires divalent metal ion:increasing the Mg2+ concentration from 0.1 to 1 mMstimulates cleavage, although a 10 mM concentrationsuppresses it (Figure 8C). The same results were obtainedwhen Ca2+ was substituted for Mg2+ (Figure 8C). Thelow-level cleavage observed in the absence of addeddivalent metal ion is probably due to endogeneous metalion, since it is suppressed by adding excess EDTA priorto ascorbate and hydrogen peroxide addition (data notshown). The data indicate that enhanced cleavage derivesfrom an ascorbate/hydrogen peroxide-dependent process,occurring in the metalloenzyme-RNA complex. Theinvolvement of a metalloenzyme-induced RNA conforma-tional change in promoting cleavage remains a possibility.The ascorbate/peroxide-dependent cleavage pattern of

Ri1.[WC-L] RNA is summarized in Figure 9C. Thestrong cleavage sites are symmetrically arrayed about thecanonical RNase III processing sites, while several weakersites occur at the top of the stem. The proximity of themajor sites of cleavage to the RNase III scissile bondssuggests some type of involvement of the enzyme catalyticsites, in which presumably reside one or more divalentmetal ions. The weaker sites may reflect other specificallybound metal ions. The reaction could involve cleavageof the phosphodiester linkages by activated vicinal 2'-hydroxyl groups or may result from ribose attack byhydroxyl radicals, locally generated by a novel pathway.However, this latter mechanism is problematic, sinceneither Mg2+ nor Ca2+ readily participate in oxidation/reduction reactions and under certain conditions Mg2+can inhibit hydroxyl radical production (Hertzberg andDervan, 1984). Although the exact source of this reactionremains to be identified, it may occur in other metallo-enzyme-nucleic acid complexes and provide a probe ofprotein-nucleic acid interactions.

DiscussionThis study describes the use of ethylation interference andhydroxyl radical footprinting to identify substrate ribose-phosphate backbone sites that interact with RNase III.The experiments took advantage of the biochemicalproperties of the rmc[E1 17A] and rnc[EI 17K] RNase IIImutants, which bind substrate but cannot catalyzecleavage. The two mutants otherwise closely resemblewild-type RNase III. First, both proteins exhibit dimericbehavior, as shown by gel filtration and chemical cross-linking. Second, the catalytic inactivity does not reflectnon-productive binding, as (i) the mutant proteins do notbind substrate more tightly than wild-type enzyme underoptimal (+Mg2+) processing conditions and (ii) wheredirect comparison was possible, the interference and

1429


footprint patterns are essentially the same for mutant andwild-type proteins.

Ethylation of specific phosphodiester linkages in Rl.1RNA and Ri.1[WC-L] RNA interferes with RNase IIIbinding and specific ribose residues are protected byRNase III from hydroxyl radicals. The physical proximitiesof the pro-interfering phosphodiesters and protected ribosegroups indicate that RNase III contacts substrate at thesesites. Moreover, the spatial distribution of the sites indicatethat the RNase 111-substrate interaction spans approxim-ately two helical turns and is essentially symmetric aboutthe scissile bonds. These findings rationalize the occur-rence of an ~20 bp (two-turn) dsRNA element in RNaseIII processing signals which contain centrally locatedcleavage sites (Dunn, 1982; Robertson, 1982). The inter-ference and footprinting results predict that protein-RNAcontacts would be lost if the upper stem suffers deletionsof >2 bp. In fact, while increasing the length of the Rl.lRNA upper stem does not alter reactivity or selectivity,shortening the stem by >2 bp significantly reduces thecleavage rate (Chelladurai et al., 1993).A model for the RNase III-processing substrate inter-

action can be proposed on the basis of these and otherdata. First, RNase III contacts substrates on both faces ofthe double helix, as there occurs significant protection orinterference at one-half and one helical turn in eitherdirection from the scissile bonds. Second, since the scissilebonds in dsRNA are on the same side of the A-helix, andacross the minor groove (Bram et al., 1980), RNase IIImaintains that interaction with the minor groove at sitesdistal to the scissile bonds.The proximity of the scissile bonds suggests that the

catalytic sites of each subunit are in close proximity. Thiswould explain the relatively enhanced reactivity of thernc[E117K] protein to DSS, which has an 11.4 A cross-linking arm. Perhaps the catalytic sites (and the reactiveLys 1 7 residues) are within this distance of each other.

Third, each subunit recognizes a processing substrate'half-site' on either side of the scissile bond(s), consistingof one helical turn (11 bp). Productive binding wouldplace a scissile bond in each of the two catalytic sites. Itwould be predicted that RNase III may cleave smaller(<20 bp) substrates by binding to a single half-site,provided there is enough additional dsRNA to place aphosphodiester linkage in a catalytic site.The RNase III subunit contains a dsRNA binding motif

(dsRBM). Taking into account the dimeric nature of RNaseIII and the (quasi) 2-fold helical symmetry of its substrates,the interference and protection patterns would suggest thateach dsRBM interacts with the minor groove of a substratehalf-site. However, the footprinting and interference resultscannot distinguish between dsRBM-RNA contacts andthose made with the remaining N-terminal two-thirds ofRNase III, which includes the catalytic site. Studies onan isolated dsRBM of the mammalian dsRNA-activatedprotein kinase, DAI (PKR) indicate a dsRBM interactionprimarily with the minor groove, within one helical turnof dsRNA (Clarke and Mathews, 1995). An NMR solutionstructure for the dsRBM of RNase III has been reportedrecently (Kharrat et al., 1995), as well as one of the threedsRBMs of Drosophila staufen protein (Bycroft et al.,1995). In the absence of direct structural information onthe dsRBM-dsRNA interaction, the compact, ellipsoidal

aX PI 2P3a2 tertiary fold exhibited by the dsRBM suggestsan interaction occurring within one helical turn, withphosphodiester oxygens engaging in ionic bonds withconserved basic residues at or near the N-terminus of a2(Bycroft et al., 1995; Kharrat et al., 1995). 2'-Hydroxylgroups may also be essential for recognition (Nicholson,1992).The more subtle differences in the Rl.1 RNA and

Rl.l[WC-L] RNA interference and protection patternsprovide additional insight on the structure-reactivity rela-tionships in RNase III processing signals and in particularthe role of internal loops on processing reactivity. The 5'-and 3'-end-proximal segments of the Rl.1 RNA internalloop occupy different environments in the enzyme-sub-strate complex. Specifically, RI. 1 RNA exhibits more pro-interfering phosphodiesters than Rl.1[WC-L] RNA, withthe additional positions mapping within or next to theinternal loop 5'-end-proximal segment. This segment isnot as well protected from hydroxyl radical attack. Anasymmetic internal loop placed in the RNase III activecenter may be anticipated to undergo single cleavage, dueto the non-productive interaction of the 5'-end-proximalinternal loop segment with a catalytic site. Cleavage ofthe scissile bond in the 3'-end-proximal segment wouldbe followed by product release. In contrast, both strandsof dsRNA would be accessible to a catalytic site, allowingcoordinate double cleavage.

This model does not yet include a role for base-pairsequence in recognition, since hydroxyl radical foot-printing and phosphate ethylation interference assays can-not reveal whether RNase III directly contacts specificbase-pair groups. However, the pro-interfering phospho-diesters and protected ribose groups partially overlap theconserved bp of a proposed consensus RNase III pro-cessing substrate (Krinke and Wulff, 1990). Also, specificbase-pair substitution in an RI. 1 RNA variant stronglyinhibit enzymatic cleavage in vitro (K.Zhang and A.W.Nicholson, unpublished). If RNase III does interact withspecific bp groups, the interference and footprinting resultssuggest that the interaction would occur in the minorgroove.

Divalent metal ions play an essential role in catalysisof RNase III processing (Li et al., 1993), but theirinvolvement in substrate recognition is less clear. Thepresence of Mg2+ or Ca2+ cannot overcome the inhibitoryeffect of ethylation of specific phosphodiester linkages,but confers stronger protection against hydroxyl radicalswithout altering the pattern. We conclude that divalentmetal ion does not play an essential role in substraterecognition, apart from enhancing substrate binding. SinceCa2+ neither supports catalysis nor inhibits Mg2+-dependent cleavage (Li et al., 1993), substrate bindingmay be enhanced by metal ions separate from thoseserving as catalytic cofactors.The Kd values for the RNase III-substrate complex are

smaller than the corresponding Km (Mg2+) values forthe processing reactions: the Km for Ri.1[WC-L] RNAcleavage (37 nM) (Chelladurai et al., 1993) is ~20-foldgreater than the Kd (Ca2+) value, and the Km for Rl.1RNA cleavage (330 nM) (Chelladurai et al., 1993) is 180-fold greater than the corresponding Kd (Ca>) value. SinceCa2+ is equivalent to Mg2+ in enhancing substrate binding(Table I) and given that the Km value reflects all events

1430


occurring in the catalytically active enzyme-substrate-Mg2+ complex (Fersht, 1985), perhaps a portion of thebinding energy is used up to attain the transition state inthe RNase III-substrate-Mg'+ complex. If so, this wouldbe reflected in the Km>Kd relationship.

Changing Glu117 to lysine or alanine in RNase IIIuncouples substrate binding from cleavage. Carboxylategroups are essential participants in the active sitechemistries of many phosphodiesterases (e.g. seeKatayanagi et al., 1990; Yang et al., 1990; Beese andSteitz, 1991; Winkler et al., 1993). The involvement ofone or more carboxylate groups in the RNase III catalyticmechanism is also indicated by the rapid, carbodiimide-dependent inactivation of phosphodiesterase activity withnegligible concomitant inhibition of substrate binding(H.Li and A.W.Nicholson, unpublished). Does Glu 117directly participate in the catalytic mechanism? Sequencealignment of RNase III-related enzymes has shown thatGlu117 is an invariant residue (lino et al., 1991; Zuberet al., 1994). Assuming a conserved catalytic mechanism,then active site residues, perhaps including Glu 117, wouldalso be conserved. However, definition of the catalyticmechanism and participatory amino acid residues awaitsfurther biochemical studies.

Materials and methodsMaterialsChemicals were of reagent grade quality. Water was deionized anddistilled. Ammonium iron(II) hexahydrate was from Aldrich (Milwaukee,WI). The cross-linking reagents DSS and DST were purchased fromPierce (Rockford, IL). ENU and sodium ascorbate were from Sigma (StLouis. MO), as was bulk-stripped Ecoli tRNA, which was furtherpurified by repeated phenol extraction. Hydrogen peroxide (30%) wasfrom Fisher. Radiolabeled nucleotides [a-32P]UTP (3000 Ci/mmol) and[y-32P]ATP (3000 Ci/mmol) were from Dupont-NEN (Boston, MA).VentR DNA polymerase and NdeI and BamHI restriction enzymeswere obtained from New England Biolabs (Beverly, MA), while T4polynucleotide kinase was from Promega (Madison, WI). Calf intestinealkaline phosphatase was from Boehringer-Mannheim (Indianopolis, IN).Sequenase (version 2.0) was from United States Biochemical Corporation(Cleveland, OH). Enzymes were used with the supplied buffers. RNaseIII was purified from an overexpressing Ecoli strain as described (Liet al., 1993). Enzyme concentrations (reported as dimer form) andspecific activities were determined as described (Li et al., 1993). T7RNA polymerase was purified as described (Grodberg and Dunn, 1990).Escherichia coli strain DH5a was from Gibco-BRL (Grand Island, NY).Plasmid pET-I la and E.coli strain BL21(DE3) were from Novagen(Madison, WI). The RNase 1II- strain, BL21(DE3)rnclO5 (lino et al.,1991) was a kind gift from M.Yamamoto. DNA oligonucleotides weresynthesized by Midland Certified Reagent Company (Midland, TX)and further purified by polyacrylamide-7 M urea gel electrophoresis(Chelladurai et al., 1991).

Construction of RNase 111 mutantsMutations were introduced into the RNase III (rnc) gene by two-stepasymmetric PCR using a single mutagenic primer (Perrin and Gilliland,1990). The mutagenic primer used to create the E117K mutation was:5'-ATTAATGCTTTGACGGTGTCG-3' (339), while the primer for theEl 17A mutation was 5'-ATTAATGCTGCGACGGTGTCG-3' (339) (thebold-faced, italicized nucleotides are the mutagenic residues, and thenumber in parentheses indicates the position in the rnc gene of theprimer 3' nt). The primers which encode the 5' or 3' end of the rncgene (rnc 5' and rnc 3' primers) are described elsewhere (Li et al.,1993). First-stage asymmetric PCR involved the mutagenic oligo (45pmol), rnc 5' primer (30 pmol), template (50 fmol pET-rnc) (Li et al.,1993), VentR DNA polymerase (2.5 units) and the four dNTPs (200 ,uMeach) in a 100 RI volume. Thirty cycles were performed (94°C, I min;55°C, I min; 72°C, 3 min) using a Perkin Elmer-Cetus Model 9600GeneAmp PCR System. The 365 bp PCR product was purified by

agarose gel electrophoresis. Second-stage PCR included: rtmc 3' primer(18 pmol), first-stage PCR DNA product (18 pmol) and template (0.1fmol pET-rnc). using the reaction conditions described above. The 697bp product was purified by agarose gel electrophoresis, cleaved withNdel and BamnHI, gel-purified, then ligated to pET-I Ia which had beencut by Ndel and BamnHI, treated with calf alkaline phosphatase and gelpurified. E.coli DH5a cells were electroporated using ligated DNA, andrecombinant plasmids identified by restriction analysis. Mutations wereverified by dideoxy sequencing using the rmic 5' and rmic 3' oligonucleo-tides as primers. No additional adventitous mutations were detected.To overexpress protein. plasmids pET-rnc(El 17K) and pET-

rnc(El 17A) were electroporated into BL21(DE3)rnclO5 cells. Therncl05 allele was required to avoid contamination by endogeneous wild-type RNase III activity. Since the rtc 1O5 and rnc70 mutations occur atseparate positions in the rnc gene, a concern was possible homologousrecombination, which would yield the wild-type gene [a recA- versionof BL21(DE3)rnclO5 provided poor expression of RNase III]. Therefore,plasmid-containing BL21(DE3)rnclO5 cell cultures grown in LB medium(50 1g/ml ampicillin) were monitored for fast-growing colonies (presum-ably rnc+ revertants). Only those cultures which lacked fast-growingcolonies were used for protein isolation. Proteins were purified from celllysates by ammonium sulfate treatment of the inclusion body, followedby poly(I)-poly(C)-agarose chromatography and SuperDex 75 gel filtra-tion (Li et al., 1993). To avoid cross-contamination, separate poly(I)-poly(C)-agarose columns were used to purify mutant and wild-typeproteins.

Synthesis of RNase 111 processing substratesInternally labeled 32P-Iabeled RNAs were prepared by in lvitro transcrip-tion of DNA oligonucleotides using T7 RNA polymerase in the presenceof rATP, rCTP, rGTP and [cx-32P]UTP (500 Ci/mol) (Chelladurai et al.,1991). The amounts of 32P-labeled RNA used in the reactions werecalculated from [x-32P]UTP specific activity and number of U residues.5'-32P-labeled RNAs were prepared by treatment of dephosphorylatedtranscripts with [y-32PIATP (3000 Ci/mmol) in the presence of T4polynucleotide kinase. RNAs were purified by gel electrophoresis(Chelladurai et al., 1991).

RNase 111 processing assaysIn vitro RNase III processing reactions were carried out essentially asdescribed, using a potassium glutamate-based buffer (Li et al., 1993).Reaction products were separated by electrophoresis in polyacrylamidegels containing 7 M urea in TBE buffer and analyzed by autoradiography.

Gel electrophoretic mobility shift assaysGel mobility shift assays were carried out as described (Chelladuraiet al., 1993), using 5'-32P-labeled RNA and purified protein. Bindingreactions (20 Il) also contained 160 mM KCI, 30 mM Tris-HCl (pH7.5), 0.1 mM DTT and 0. 1 mM EDTA (or 5 mM, when Mg2+was omitted) and 10% glycerol. Other specific components and theirconcentrations are given in the legends to Figures 4 and 5. Reactionswere incubated at room temperature for 10 min, cooled on ice, thenelectrophoresed (10 V/cm, 5-6°) in 8% polyacrylamide gels (80:1 ratioacrylamide to bisacrylamide). Quantitation of the gel shift assays wasperformed using an AMBIS radioanalytic imaging system (see Table I).

Ethylation interference assaysRNA ethylation reactions were carried out in 200 gl volumes, whichincluded 5'-32P-labeled RNA (2X 106 d.p.m.; 0.3 pmol 5'-32P-phosphate)and 10 jg tRNA in 50 mM sodium cacodylate (pH 7.5), 1 mM EDTA.A saturated solution of ENU in ethanol (40 jl) was added to initiate thereaction and modification was allowed to proceed for 90 min at 37°C.Radioanalytic imaging and quantitation of the gel electrophoretic patternof the Tris base-treated (see below) ethylated RNA indicated that -20%of the RNA had been modified. Reactions were quenched and the RNAprecipitated by adding 40 ji of 3 M sodium acetate (pH 5.2) and 3 volof 95% ethanol. The recovered RNA was resuspended in H,O and addedto an RNase III binding reaction (see above). Conditions were chosento maximize observation of an inhibitory effect of ethylation on RNaseIII binding. Thus, an amount of RNase III was added to provideapproximately equal amounts of free and bound RNA, as determined bynon-denaturing gel electrophoresis. Binding reactions were incubated atroom temperature for 10 min, then electrophoresed (10 V/cm, 5-.6C)in an 8% non-denaturing polyacrylamide gel. The free and bound RNAswere extracted from crushed gel slices by phenol treatment and ethanolprecipitation, then incubated in 0.1 M Tris-HCI (pH 9.0) (10 ,ul) for5 min at 50°C. The RNAs were precipitated by adding 3 M sodium

1431


acetate (pH 5.2) (4 ,ul) and 95% ethanol (40 ,ul), then washed twice withcold 95% ethanol, dried and resuspended in gel loading buffer. Equalamounts of radioactivity were electrophoresed (33-36 V/cm) in a 12 or15% polyacrylamide sequencing gel containing 0.5X TBE buffer and7 M urea. Gels were autoradiographed at -70°C using intensifyingscreens. Additional relevant experimental details are given in the legendto Figure 6.

Hydroxyl radical footprinting assaysHydroxyl radical footprinting was carried out as described (Latham andCech, 1989) with several modifications. Since glycerol quenches thehydroxyl radical reaction, it was removed from the RNase III preparationsby gel filtration on SuperDex 75 using a buffer containing 30 mMMOPS (pH 7.5), 250 mM NaCl, 0.1 mM EDTA and 0.1 mM DTT. Theglycerol-free enzyme was stored at -70°C prior to use. The hydroxylradical generating mix was prepared by combining a solution (10 ,ul) of10 mM ferrous ammonium sulfate and 20 mM Na3-EDTA with 10 mMsodium ascorbate (10 ,ul) and 0.6% H202 (10 gl). Na3-EDTA was usedto neutralize more effectively the acidic ferrous ammonium sulfatesolution. The mix was immediately added to the protein-RNA bindingreaction (40 gl), which had been preincubated at room temperature for10 min. Binding reactions contained: 5'-32P-labeled RNA (105 d.p.m.,15 fmol), RNase III (20 pmol) and Ecoli tRNA (3 jg) in a buffercontaining 160 mM KCI, 20 mM MOPS (pH 7.5), 0.1 mM EDTA and0.1 mM DTT. Control reactions omitted RNase III. Additional specificsof the assay are given in the legend to Figure 8. Following reaction for1.5 min at room temperature, the reactions were quenched by adding0.1 M thiourea (8 gl). RNA was treated with phenol-chloroform (1:1v/v) and ethanol-precipitated. RNA was resuspended in 1.5 ,l H20 and1.5 ,ul sequencing dyes were then added. Samples were electrophoresed(33-36 V/cm) in a 12 or 15% polyacrylamide sequencing gel containing0.5X TBE buffer and 7 M urea. Autoradiography was carried out usingintensifying screens.

AcknowledgementsOne of the authors (A.W.N.) would like to thank Dr David Draper fora helpful discussion on RNA conformation and gel mobility. Bothauthors thank other members of the lab for their advice and interest inthese studies. This project is supported by NIH grant GM41283.

ReferencesBeese,L.S. and Steitz,T.A. (1991). Structural basis for the 3'-5'

exonuclease activity of Escherichia coli DNA polymerase I: a twometal ion mechanism. EMBO J., 10, 25-33.

Bram,R.J., Young,R.A. and Steitz,J.A. (1980) The ribonuclease III siteflanking 23S sequences in the 30S ribosomal precursor RNA of Ecoli.Cell, 19, 393-401.

Bycroft,M., Grunert,S., Murzin,A.G., Proctor,M. and St Johnston,D.(1995) NMR solution structure of a dsRNA binding domain fromDrosophila staufen protein reveals homology to the N-terminal domainof ribosomal protein S5. EMBO J., 14, 3563-3571.

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Received oni Mayv 3. 1995: r-evised oni November 10, 1995

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interference and hydroxyl radical footprinting using

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