MOLECULAR AND CELLULAR BIOLOGY, Sept. 1993, p. 5149-51580270-7306/93/095149-10$02.OO/OCopyright ©) 1993, American Society for Microbiology

Proteolytic Footprinting of Transcription Factor TFIIIAReveals Different Tightly Binding Sites for

5S RNA and 5S DNADANIEL F. BOGENHAGEN

Department ofPharmacological Sciences, State University ofNew York at Stony Brook,Stony Brook, New York 11794-8651

Received 29 March 1993/Returned for modification 4 May 1993/Accepted 1 June 1993

Transcription factor MIIA (TFIA) employs an array of nine N-terminal zinc fingers to bind specifically toboth 5S RNA and 5S DNA. The binding of TFIIIA to 5S RNA and 5S DNA was studied by using a proteasefootprinting technique. Brief treatment of free or bound TFIIIA with trypsin or chymotrypsin generatedfragments which were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Fragmentsretaining the N terminus of TFIIIA were identified by immunoblotting with an antibody directed against theN terminus of TFIIIA. Proteolytic footprinting of TFIIIA complexed with 5S DNA derivatives reinforced otherevidence that the three N-terminal zinc fingers ofTFIIIA bind most tightly to 5S DNA. Proteolytic footprintingof TFIIIA in reconstituted 7S ribonucleoprotein particles revealed different patterns of trypsin sensitivity forTFIA bound to oocyte versus somatic 5S RNA. Trypsin cleaved TFIA between zinc fingers 3 and 4 more

readily when the protein was bound to somatic 5S RNA than when it was bound to oocyte 5S RNA. A trypticfragment of TFIIIA containing zinc fingers 4 through 7 remained tightly associated with somatic SS RNA. Zincfingers 4 through 7 may represent a tightly binding site for 5S RNA in the same sense that fingers 1 through3 represent a tightly binding site for 5S DNA.

TFIIIA is a unique example of a zinc finger transcriptionfactor that binds specifically to 5S RNA as well as to 5SDNA (20, 30). TFIIIA is typically prepared from an abun-dant 7S storage particle from immature Xenopus laevisovaries. Smith et al. (37) showed that TFIIIA within the 7Sparticle could be cleaved with proteases such as papain andtrypsin to yield fragments of the protein that retained theability to bind to the intragenic control region (ICR) of the 5SRNA gene. A 30-kDa fragment that contains nine zincfingers provided essentially the same DNase I footprint over

residues 46 to 95 of the 5S RNA gene obtained with intactTFIIIA. A 20-kDa fragment that contains the N-terminal 220residues of TFIIIA (seven fingers) protected only the distalportion of the ICR of the 5S RNA gene from digestion byDNase I. This work provided the first clear evidence thatTFIIIA binds to the ICR in an extended conformation withzinc fingers 1 through 9 aligned from the 3' to the 5' end ofthe ICR. This alignment leaves the carboxyl-terminal non-

zinc finger domain of TFIIIA near the 5' portion of the ICR,where it appears to interact with other transcription factors(19, 37). Two groups have recently shown that amino-terminal fragments of TFIIIA containing as few as three zincfingers bind tightly to the distal portion of the ICR (5, 25).Indeed, fingers 1 through 3 bind to gene residues 77 through92 nearly as tightly as intact TFIIIA binds to the completeICR. This implies that specific contacts of fingers 4 through9 of TFIIIA with gene sequences located 5' to this tightlybinding site are not as energetically favorable. Currentmodels suggest that fingers 4 and 6 may not make tightcontacts in the major groove of DNA (6, 11, 18). However,crystallographic or nuclear magnetic resonance analyses ofTFIIIA-5S DNA structures have not been presented to date.The binding of TFIIIA to 5S RNA is not as well under-

stood as its binding to 5S DNA is. Naturally occurring 7S

particles can contain either somatic or oocyte 5S RNA.These two forms of 5S RNA differ somewhat in sequence butare thought to have similar structures. Although the tertiarystructure of 5S RNA has not been determined, secondarystructure models feature three helical stems that converge ina central crux (40). TFIIIA appears to contact all threebase-paired stems of 5S RNA, with the most importantcontacts near the convergence of these three RNA helices(4, 38). However, TFIIIA tolerates sequence substitutionsthat leave the base-paired structure intact (38, 41).

In this report, I present a proteolytic footprinting methodto study the interaction of TFIIIA with 5S RNA and 5SDNA. This method is patterned after nuclease footprintingprocedures that are extremely useful in identifying regions ofDNA protected from nuclease attack by specific bindingproteins.

MATERIALS AND METHODS

5S DNA and 5S RNA. The sequences of three restrictionfragments related to the ICR of the X. borealis somatic (Xbs1) 5S RNA gene (31) used in this study are shown in Fig. 4C.The 65-mer wild-type sequence was a gift from LouiseFairall. The two fragments containing foreign sequences inplace of the 5' portions of the ICR were synthesized as

oligonucleotides and cloned in pSK vectors (Stratagene) as

tandem repeats. The inserts were excised by cleaving withEcoRV and were purified by high-pressure liquid chroma-tography anion-exchange chromatography on a GenPak Faxcolumn (Waters). 5S RNA was prepared by runoff transcrip-tion in vitro by T7 RNA polymerase of a cloned templatecontaining the Xbs 1 5S RNA gene as described by Bogen-hagen and Sands (4). An oocyte RNA expression constructwas prepared by substituting the EaeI-FokI fragment of theXbs 1 gene with the corresponding portion of the X. laevis

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oocyte SS RNA gene (derived from Xlo 31 [28]). RNA was

purified by chromatography on a GenPak Fax column,extracted with phenol-chloroform followed by ether, andprecipitated with ethanol. RNA was resuspended in TEbuffer (10 mM Tris [pH 8], 1 mM EDTA), quantified bymeasurement ofA260, and stored frozen at a concentration ofapproximately 20 ,uM.

7S particles and TFIIIA. 7S particles were prepared fromimmature X. laevis ovaries as described by Hanas et al. (16).TFIIIA was prepared from purified 7S particles by using one

round of ammonium sulfate precipitation to remove most 55

RNA (35) followed by chromatography on Bio Rex 70(Bio-Rad Laboratories) (37). This combination of stepsavoids RNase treatment of the particles. TFIIIA used inbinding reactions was diluted to S or 10 p,M protein inbinding buffer containing 0.05% Triton X-100. A seven-fingerfragment of TFIIIA used as a mobility marker in some

experiments was prepared as the product of a clone in whichT7 RNA polymerase was used to express TFIIIA. Thecarboxyl terminus of the product was determined by a

nonsense mutation (UAU- UAA) in codon 221 of TFIIIAinserted by using polymerase chain reaction amplification.The recombinant 220-amino-acid protein was purified as

described by Del Rio and Setzer (10) and shown (2a) tocomigrate on gels with the 20-kDa fragment of TFIIIAstudied by Smith et al. (37).

TFIIIA-nucleic acid binding reactions and native agarose

gel electrophoresis of complexes. TFIIIA was mixed withnucleic acids at concentrations of 0.5 to 1 ,uM in a buffercontaining 60 mM KCl, 2mM MgCl2, 2 mM dithiothreitol, 10,M zinc acetate, 8% glycerol, and 10 mM N-2-hydroxyeth-ylpiperazine-N'-2-ethanesulfonic acid (HEPES; pH 7.5).Binding reaction mixtures included a final concentration of0.02% Triton X-100. Reactions including RNA or 7S parti-cles were supplemented with RNasin (Promega) at a finalconcentration of 1 U/,u. TFIIIA was typically added as thelast ingredient to binding reaction mixtures so that nucleicacid would be available for binding as soon as the proteinwas diluted. In experiments in which saturation of TFIIIAwith nucleic acid was desired, protein was mixed with a

1.5-fold molar excess of nucleic acid. Binding reactionmixtures were incubated for 15 min at 21°C before analysisby proteolytic footprinting or electrophoresis. Protein-nu-cleic acid complexes were subjected to electrophoresis on

1.2% agarose minigels containing 0.02% Triton X-100 as

described by Bogenhagen and Sands (4).Peptide synthesis, antipeptide antisera, and immunoblot-

ting. The peptide MGEKALPVVYKRYIC was synthesizedby the Center for Analysis and Synthesis of Macromoleculesfacility at the State University of New York-Stony Brook,using standard solid-state peptide synthesis on an AppliedBiosystems peptide synthesizer. The peptide was coupled tokeyhole limpet hemocyanin as described by Lerner et al. (24)and injected into rabbits to obtain polyclonal antisera. Tworabbits were injected and provided comparable responses,

with positive reactions at a 1:20,000 dilution in immunoblotsof intact TFIIIA. Proteins were subjected to electrophoresisin sodium dodecyl sulfate (SDS)-containing 12 or 12 to 18%gradient polyacrylamide gels in the Tris-glycine-HCl buffersystem (23). Proteins were electroblotted onto Immobilonpolyvinylidene difluoride membranes. Blots were incubatedat room temperature in phosphate-buffered saline containing0.05% Tween 20 first with a 1:10,000 dilution of antipeptideantibody and then with a 1:5,000 dilution of affinity-purifiedgoat anti-rabbit immunoglobulin G coupled to calf intestinalalkaline phosphatase (Boehringer Mannheim). Immunoreac-

tive proteins were detected by development of the blots witha colorimetric reaction using p-nitroblue tetrazolium chlo-ride and 5-bromo-4-chloro-3-indolylphosphate (Kirkegaard& Perry Laboratories).

Proteolytic footprinting. TFIIIA diluted into binding bufferin the presence or absence of nucleic acids was subjected tolimited proteolysis with modified trypsin (Promega) or withchymotrypsin (Boehringer Mannheim). Unless otherwisenoted, standard reaction conditions were used with a finalconcentration of 10 ,ug of trypsin per ml or 5 ,ug of chymo-trypsin per ml to treat 4- to 8-pmol quantities of TFIIIA in10-pl volumes for 1 min at 21°C. In some experiments, thequantities of protease used varied from 0.5 to 5 times thestandard concentrations and the times of incubation wereextended to as long as 3 min. Protease treatment wasstopped by adding the sample to an equal volume of SDSstop buffer [20 mM dithiothreitol, 62.5 mM Tris-HCl (pH6.8), 25 mM EDTA, 1 mM 4-(2-aminoethyl)benzenesulfo-nylfluoride (AEBSF), 10% glycerol, 2% SDS, 0.03% bromo-phenol blue] held in a water bath at 85°C. Addition of thisSDS stop buffer at room temperature instead of 85°C did notterminate proteolysis as effectively. When all samples from aseries of footprinting reactions were collected, the completeset of tubes was moved to a boiling water bath for 2 min. Allsamples were treated with 0.1 M iodoacetamide for 30 minbefore being loaded onto gels. TFIIIA was fragmented ineither of two ways to provide homologous mobility markersfor gel electrophoresis. CNBr (Sigma) and 2-(2'-nitrophenyl-sulfonyl)-3-methyl-3-bromoindolenine (BNPS)-skatole (Pierce)were used to cleave TFIIIA at methionine and tryptophanresidues, respectively, as described previously (1). Alterna-tively, a 5-pg sample of TFIIIA was denatured by boiling in0.2% SDS in the presence of 5 mM EDTA and treated with0.4 ,g of Asp-N protease (Boehringer Mannheim). To obtaina heterogeneous set of fragments, aliquots of the digestionreaction mixture were removed after 1, 2, 5, 15, and 30 minand added to a single tube containing SDS stop buffer at80°C. After completion of the reaction, fragments weretreated with 0.1 M iodoacetamide and stored in aliquots at-20°C. To aid in determining protease cleavage sites, immu-noblots were scanned with an LKB Ultroscan XL laserdensitometer to measure the relative mobilities of proteo-lytic fragments.DEAE-Sephacel chromatography. DEAE-Sephacel (Phar-

macia) was pretreated with 2 mg of bovine serum albumin(BSA) in binding buffer containing 50 mM NaCl in place ofKCl and washed with binding buffer containing 0.5 M NaCl.Columns of 80-pl volume were packed in disposable pipettetips plugged with siliconized glass wool. Binding reactionmixtures were scaled up to contain 100 pmol of TFIIIA and150 pmol of nucleic acid in a volume of 100 pl and treatedwith trypsin under standard conditions. After 1 min oftrypsin treatment, 1 ,ul of 0.1 M AEBSF was added and thesamples were loaded onto DEAE-Sephacel columns. Col-umns were loaded and washed with 3 volumes of bindingbuffer with slight pressure from a Pipetman P-200 pipettor(Rainin). Proteins bound to the resin were eluted with 2column volumes of binding buffer containing 0.5 M NaCl. Incontrol experiments, aliquots were subjected to native aga-rose gel electrophoresis to show that complexes remainedintact (data not shown). In most cases, the eluate was mixedwith an equal volume of SDS stop buffer and boiled. Proteinswere concentrated by precipitation with 10% trichloroaceticacid and analyzed by SDS-polyacrylamide gel electrophore-sis (PAGE) essentially as for direct proteolytic footprintingreactions. When proteins eluted from DEAE-Sephacel were

MOL. CELL. BIOL.

PROTEOLYTIC FOOTPRINTING OF TFIIIA 5151

AA No. Finger No.M G E KA L

P V V Y K R Y I C S F A D C G A A Y N K N W K L Q A H L C K H 37

T G E K P F P C K E E G C E K G F T S L H H L T R H S L T H 67

T G E K N F T C D S D G C D L R F T T K A N M K K H F N R F H 98

N I K I C V Y V C H F E N C G K A F K K H N Q L K V H Q F S H 129

T Q Q L P Y E C P H E G C D K R F S L P S R L K R H E K V H 159

A G Y P C K K D D S C S F V G K T W T L Y L K H V A E C H 188

Q D L A V C D V C N R K F R H K D Y L R D H Q K T H 214

E K E R T V Y L C P R D G C D R S Y T T A F N L R S H I Q S F H 246

E E Q R P F V C E H A G C G K C F A M K K S L E R H S V V H 276

2

3

4

5

6

7

8

9

D P E K R K L K E K C P R P K R S L A S R L T G Y I P P K S K E K N 310A S V S G T E K T D S L V K N K P S G T E T N G S L V L D K L T I Q 344

M90

v

M265

4,344

265

90

254 CNBr17579

w w28 177

177

28

316 LBNPS-Skatole167

149

FIG. 1. Location of the N-terminal epitope with respect to chemical cleavage sites in TFIIIA. The amino acid (AA) sequence of TFIIIA(14) is shown at the top, with the Cys and His residues of the zinc fingers aligned and in boldface type. Lys and Arg residues are also shownin boldface. Predicted partial and complete digest products are shown below. Those fragments that retain the N terminus (thick bar) areexpected to react with antiserum on immunoblots. Sizes of fragments are given as the number of amino acid residues.

prepared for sequencing, the protein recovered from thecolumn from an initial 100-pmol sample of TFIIIA incubatedwith 5S RNA was subjected to electrophoresis in a single gellane. Proteins were electroblotted to Immobilon polyvinyl-idene difluoride membranes and detected by briefly stainingthe blot with Coomassie blue. The excised band of interestwas sequenced in an Applied Biosystems model 475A pro-tein sequencer.

RESULTS

Development of an indirect end-labeling procedure to mon-itor proteolysis. The proteolytic footprinting method is de-signed to resemble DNase I footprinting (13) procedures inwhich products of nuclease digestion are detected with anend-labeled probe (15, 27). To provide an end-labeling probefor TFIIIA, I used an antiserum directed against the 15 N-terminal resides of TFIIIA (14), MGEKALPVVYKRYIC.This antipeptide antiserum reacted strongly with TFIIIA onimmunoblots at dilutions of approximately 1:20,000. Todetermine whether the antiserum reacted selectively with

the N terminus of TFIIIA, samples of TFIIIA were partiallydigested with either CNBr, which cleaves at methionine (M)residues, or BNPS-skatole, which cleaves at tryptophan (W)residues (Fig. 1). The digestion products were resolved bySDS-PAGE and detected either by staining with Coomassieblue (Fig. 2A) or by immunoblotting with the antipeptideantibody (Fig. 2B). The antipeptide antibody reacts selec-tively with fragments that retain the N terminus of TFIIIA.These chemical degradation products or a ladder of frag-ments produced by partial digestion of denatured TFIIIAwith sequencing-grade Asp-N protease (Fig. 2C) were usedas markers in proteolytic footprinting experiments. Thepattern of fragments revealed by immunoblotting the Asp-Ndigestion products agrees well with the known locations ofaspartic acid residues within TFIIIA. Even with denaturedprotein, the efficiency of cleavage at individual sites is notabsolutely uniform. For example, cleavage at residue 166 or276 is frequently weaker than cleavage at other sites. Theladder in Fig. 2C indicates that this method is capable ofresolving cleavage sites separated by as few as five amino

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5152 BOGENHAGEN

A B12 12

*-344-265

*; -177*-9o

C0 1 5 30 CNBr

344A" :a I1_344344~~so* ==319--"' 'sn -- 276 265_ml* jjt 15= 228,225_ 3- 208,204__ll 194,189

166- _ 4_ 142

8077,75

90

FIG. 2. Specificity of proteolytic footprinting. (A) TFIIIA wastreated with either BNPS-skatole (lane 1) or CNBr (lane 2). Result-ing fragments were resolved by SDS-PAGE and detected by stainingwith Coomassie blue. Sizes are indicated in kilodaltons. (B) Thefragments resolved by SDS-PAGE and stained in panel A weredetected by immunoblot analysis with the anti-N-terminal anti-serum. Sizes are indicated in kilodaltons. (C) A sample of TFIIIAwas denatured and treated with Asp-N protease for 1, 5, or 30 min.Reactions were stopped by boiling in SDS. Samples were analyzedby SDS-PAGE alongside a partial CNBr digest of TFIIIA, shown inthe righthand lane. Fragments retaining the N terminus were de-tected by immunoblot analysis. The numbers in the center reflect thesizes (in amino acid residues) of fragments derived by assignment ofcleavage products to the known locations of aspartic acid residues inTFIIIA.

acid residues. As expected, resolution is diminished forhigher-molecular-weight fragments.

Binding to 5S DNA or 5S RNA alters the accessibility ofTFIIIA to trypsin. Two conditions must be satisfied to allowfurther development of the protein footprinting method.First, proteases that will cleave TFIIIA at a number of sitesduring a brief incubation must be identified. By analogy withthe strategy for DNase I footprinting, it was considered thatbrief incubations with protease would be unlikely to disruptprotein-nucleic acid complexes. Results that validate thisassumption are presented below. Second, to compare cleav-age sites within free TFIIIA with those within TFIIIA boundto nucleic acid, the protein preparation must have goodbiological activity. Any significant fraction of TFIIIA notbound to nucleic acid would presumably provide a back-ground cleavage pattern characteristic of free TFIIIA.Therefore, assembly of protein-nucleic acid complexes wasdone under conditions in which complete binding of proteinis driven by a slight excess of nucleic acid.TFIIIA contains 344 amino acid residues, with 59 and 53

potential sites for cleavage by trypsin and chymotrypsin,respectively. However, these sites are not likely to becleaved at equivalent rates in the native protein. Preliminaryexperiments were tried with varied concentrations of severalproteases. The most useful cleavage reagents were judged tobe trypsin, which cleaves at lysine and arginine residues, andchymotrypsin, which cleaves at phenylalanine, tyrosine,tryptophan, leucine, and methionine residues. Cleavageswith proteinase K, papain, V8 protease, and thermolysinwere found to be less useful for brief protease treatments.These reagents showed few differences between cleavagepatterns of free and bound TFIIIA.

Figure 3 shows a comparison of the tryptic (Fig. 3A) andchymotryptic (Fig. 3B) digests of free TFIIIA, TFIIIAbound to 5S RNA in the 7S particle, and TFIIIA bound tothe ICR of the 5S RNA gene. The 7S particle is a naturallyoccurring 1:1 complex of TFIIIA and 5S RNA. The DNAcomplex was assembled in vitro by mixing TFIIIA with a1.5-fold molar excess of a short restriction fragment contain-

Al 2 3 115 6 7 T M

1- 1 2 :1 -1 : - NI

A 00

FIG. 3. Comparison of the proteolytic fragments of free TFIIIAand of TFIIIA complexed with 5S DNA or 5S RNA. Immunoblotanalyses of partial proteolytic digests of free TFIIIA (lanes 2 and 3),TFIIIA in the 7S particle (lanes 4 and 5), and TFIIIA bound to 5SDNA (lanes 6 and 7) are shown. (A) Tryptic digests. Samples inlanes 2, 4, and 6 were digested with the standard amount of trypsin;samples in lanes 3, 5, and 7 were digested with 2.5 times thestandard amount. Lanes 1 and M contain intact TFIIIA (labeled A)and endoproteinase Asp-N fragments, respectively, as described forFig. 2C. The sizes (in amino acid residues) of N-terminal fragmentsof TFIIIA are indicated on the right. Lane T contains the higherquantity of trypsin used in lanes 3, 5, and 7 and serves to show thatthe immunological reagents react to some extent with trypsin. (B)Chymotryptic digests. Samples in lanes 2, 4, and 6 were digestedwith the standard amount of chymotrypsin; samples in lanes 3, 5,and 7 were digested with 3 times the standard amount. Lanes 1 andM contain intact TFIIIA (labeled A) and endoproteinase Asp-Nfragments, respectively, as described for Fig. 2C. The sizes (inamino acid residues) of N-terminal fragments of TFIIIA are indi-cated on the right. Lane CT contains the higher quantity of chymo-trypsin used in lanes 3, 5, and 7.

ing the ICR of the 5S RNA gene. Two points can be notedbefore these cleavage patterns are analyzed in detail. First,binding of TFIIIA to either form of nucleic acid significantlychanges the pattern of fragments produced with trypsin orchymotrypsin. This finding indicates that the large majorityof TFIIIA molecules retained specific binding activity. Sec-ond, the cleavage patterns for TFIIIA in the RNA and DNAcomplexes are remarkably similar.The trypsin cleavage patterns in Fig. 3A show protection

from proteolysis at some sites and enhanced cleavage atothers. Several sites within the zinc finger domain that areaccessible to trypsin in the free protein are protected bybinding to either 5S RNA or 5S DNA. These regions includethree clusters of sites between residues 142 and 170. Thesethree sites most likely correspond to cleavage at K-144-R-145, R-151-R-154, and K-165-K-166. More extensive trypsintreatment of either RNA- or DNA-bound TFIIIA (Fig. 3A,lanes 5 and 7) produces two major products. The predomi-nant product appears to result from enhanced cleavagebetween the zinc finger domain and the carboxyl-terminalnonfinger domain, in a cluster of basic residues, K-280RK-LKEKCPRPKR-292, immediately following finger 9. Thisproduct comigrates on gels with the 30-kDa fragment ofTFIIIA generated by papain treatment of 7S particles (2a,37). The second major tryptic product in lanes 5 and 7 of Fig.3A has a size consistent with cleavage at K-216-ER-218 justafter finger 7. This site is accessible in free TFIIIA as well asin TFIIIA complexed with nucleic acid. The resulting frag-

MOL. CELL. BIOL.

PROTEOLYTIC FOOTPRINTING OF TFIIIA 5153

ment comigrates with the 20-kDa fragment produced byprolonged treatment of 7S particles with trypsin (2a, 37).The results of proteolytic footprinting with chymotrypsin

(Fig. 3B) are similar to those described above for trypsin.Cleavage sites in the zinc finger domain are protected bybinding of TFIIIA to 5S RNA or 5S DNA, and the exten-sively treated complexes (lanes 5 and 7) show abundant nine-and seven-finger products that clearly result from enhancedcleavage by chymotrypsin. The enhanced cleavage by tryp-sin or chymotrypsin following the zinc finger domain result-ing from binding to nucleic acids may reflect a change in theconformation of the protein, as if this region served as aflexible hinge. The RNA and DNA complexes permit slightlydifferent chymotrypsin cleavages in this area (compare lanes5 and 7 of Fig. 3B). However, the main conclusions providedby the proteolytic footprinting experiments shown in Fig. 3are that binding of TFIIIA to either 5S RNA or 5S DNAresults in protection of trypsin and chymotrypsin cleavagesites in the zinc finger domain and enhanced (or persistent)cleavage at sites following fingers 7 and 9.Only N-terminal domains of TFIIIA are protected by the

distal portion of the 5S DNA ICR. Figure 3 shows that bindingof TFIIIA to 5S DNA reduces the probability of cleavage bytrypsin in the zinc finger domain, although some cleavage isobserved near residue 220, in finger 7. Smith et al. (37)showed that the 30-kDa fragment, which contains the first280 to 290 residues of TFIIIA, protects the entire ICR fromDNase I. Other DNase I footprinting experiments haveshown that binding of TFIIIA to a cloned fragment of 5SDNA deleted from the 5' side to residue 68 of the ICRprotects only the remaining portion of the ICR from nucleaseattack. Unrelated sequences placed on the 5' side of thedeletion endpoint were not protected in these DNase Ifootprints (3).

Figure 4A presents the results of an experiment to deter-mine whether binding of TFIIIA to truncated versions of theICR would produce altered trypsin footprints of TFIIIA.TFIIIA was mixed with three different 65-bp DNA fragmentscontaining either the entire ICR (lane 4) or either of deletionderivatives A5' + 69 and AS' + 77, containing unrelatedsequences upstream of the deletion endpoint (lanes 5 and 6,respectively). Trypsin footprinting of TFIIIA bound to thisseries of 5' deletions of the ICR shows progressive reductionin the size of proteolytic fragments. An enhanced trypsincleavage site is observed near residue 200 when TFIIIA isbound to residues 69 to 96 of the ICR. The same fragmentand a smaller species are produced by trypsin treatment ofTFIIIA bound to the DNA fragment bearing gene residues 77to 96 (lane 6). These cleavages most likely occur in thesequences R-199-KFR-202 and K-144-R-145 in the tips offingers 7 and 5, respectively, as diagrammed in Fig. 4B.These results are consistent with the pattern of contacts ofindividual zinc fingers suggested by Hayes and Tullius (18).It appears that hypersensitive trypsin cleavage sites occurnear the boundaries of the regions complexed with DNA.How closely trypsin or another protease can cleave to theboundary of a binding site presumably depends on severalfactors, including the proximity of a sequence that repre-sents a favorable cleavage site. Although it is well knownthat trypsin can cleave small oligopeptide substrates, opti-mal efficiency of cleavage may require that a potentialcleavage site be represented in an accessible stretch ofseveral amino acids (12).An alternative method for probing complexes of TFIIIA

with 5S DNA is to characterize fragments of TFIIIA thatremain tightly associated with the DNA after proteolytic

A 1 2 3 4 5 6 M

344- _MA__O31228.225- -20.204

- 194,189142

13 28038 96

|C <|K0| | IX -n IU I,,T IC R

______ ra/6996

0 Q |1 ll 11 1[J | ;5 + 6 9

___ 20 __.j 1____ i_ll _1__+69.

40 50 60 70 80 96 100

5' -aTCTGATCTCGGAAGCCAAGCAGGGTCGGGCCTGGTTAGTACTTGGATGGGAGACCGCCTtGGAt-3'3 -tAGACTAGAGCCTTCGGTTCGTCCCAGCCCGGACCAATCATGAACCTACCCTCTGGCGGAaCCTa-5'

XBS A5'+69:

5' -atcactagagttctattccaatcacgatttaaTGGTTAGTACTTGGATGGGAGACCGCCTtGGAt-3'3 -tagtgatctcaagataaggttagtgctaaattACCAATCATGAACCTACCCTCTGGCGGAaCCTa-5'

XBS A5' -77:

5' -atcactagagttctattccaatcacgatttaagtcgacacACTTGGATGGGAGACCGCCTtGGAt-3'3' -tagtgatctcaagataaggttagtgctaaattcagctgtgTGAACCTACCCTCTGGCGGAaCCTa-5'

FIG. 4. 5' deletion of the ICR results in protection of progres-sively smaller amino-terminal fragments of TFIIIA. (A) Immunoblotof an SDS-PAGE analysis of tryptic digests of TFIIIA bound tovarious nucleic acid ligands. Lanes: 1 and M, markers of intactTFIIIA and the Asp-N ladder, respectively; 2 and 3, tryptic frag-ments of TFIIIA in the 7S particle and free TFIIIA, respectively; 4to 6, tryptic fragments of TFIIIA complexed with the intact 5S DNAICR, with A5' + 69, and with A5' + 77, respectively. Sizes areindicated in amino acid residues. (B) Diagrammatic interpretation ofthe results in panel A. Progressive 5' deletion of the ICR results inexposure of trypsin-sensitive sites closer to the N terminus ofTFIIIA. These sites are approximately identified as residues 200 and145 (arrows). Each individual zinc finger is drawn as a two-strandedi sheet coordinated through a zinc atom (small circle) to a short ahelix (thickened bar). WT, wild type. (C) Sequences of the threerestriction fragments. Uppercase letters indicate retained 5S DNAsequences.

treatment of the complexes. Anion-exchange chromatogra-phy provides a rapid method to separate free TFIIIA, whichdoes not bind to DEAE-Sephacel, from TFIIIA associatedwith DNA or RNA, which binds through the negatively

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5154 BOGENHAGEN

A wTr A 6

2 6

-A-DNA

fre e

DINA

F3 WT A69I Z 3 1 I.

A - _ - -319_" _*- 3~~' " 1 94.4 89

11

c WT A 692 3 41 N

A- -

_m4 ?o0 14 /s .,-- 4.

- E.lP

FIG. 5. Amino-terminal fragments of TFIIIA remain tightly as-sociated with 5S DNA after trypsin treatment within the TFIIIA-DNA complex. (A) Native agarose gel analysis. An ethidium bro-mide-stained agarose gel of TFIIIA-DNA complexes is shown.Lanes: 1, free 65-bp restriction fragment of the wild-type (WT) ICR;2, mixture of free and TFIIIA-bound wild-type DNA resulting fromincubation of TFIIIA with a 1.5-fold excess of the restrictionfragment shown in lane 1; 3, complexes after treatment with trypsinfor 1 min; 4 through 6, analogous to lanes 1 through 3 except that theDNA fragment used was Xbs A5' + 69. (B) SDS-PAGE analysis offragments detected by immunoblotting. Complexes of TFIIIAbound to the wild-type (WT) ICR (lane 2) or to A5' + 69 (lane 3)were treated with trypsin for 1 min and loaded onto a smallDEAE-Sephacel column as described in Materials and Methods.Proteins bound to the DEAE column were eluted with 0.5 M NaCland analyzed by SDS-PAGE and immunoblotting with the anti-amino-terminal antiserum. Lanes 1 and 4 show markers of intactTFIIIA and an amino-terminal 220-residue seven-finger fragment ofTFIIIA produced in Escherichia coli; lane L contains the Asp-Nfragment ladder described in the legend to Fig. 2. Sizes are indicatedin amino acid residues. (C) SDS-PAGE analysis of fragmentsdetected by Coomassie blue staining. Lanes 1 through 4 are asdescribed for panel B. Lane M contains low-molecular-weightprotein markers with the sizes (in kilodaltons) as indicated. Theband near the top of lanes 2 and 3 is BSA that had been retained onthe column through a pretreatment to block nonspecific binding siteson the resin.

charged nucleic acid. Chromatography on DEAE resins hasbeen a common method for preparation of 7S particles andTFIIIA (16, 30). However, this resin has not previously beenused for preparation of TFIIIA-SS DNA complexes. Anion-exchange columns can disrupt complexes of TFIIIA andnucleic acids, particularly if large bed volumes of columnmaterials are used for analysis of small quantities of com-plexes. I performed a series of control experiments to defineconditions under which free TFIIIA and trypsin would flowthrough small columns of DEAE-Sephacel while complexesof TFIIIA with nucleic acids would be retained. The appli-cation of this simple chromatographic step to analysis ofproteolytic fragments of TFIIIA bound to 5S DNA is shownin Fig. 5. To assemble TFIIIA-SS DNA complexes, 100-pmol samples of TFIIIA were mixed with a 1.5-fold excessof either the intact 5S DNA ICR or the A5' + 69 fragment

used in Fig. 4. These complexes were then treated withtrypsin for 1 min, after which the protease inhibitor AEBSFwas added. The native gel analysis in Fig. SA shows thatDNA-protein complexes were formed under these condi-tions and that proteolysis resulted in conversion of thecomplexes to a more rapidly migrating form. The trypsin-treated complexes were then loaded onto 80-,ul columns ofDEAE-Sephacel, washed with low-salt buffer, and elutedwith buffer containing 0.5 M NaCl. The proteins eluted fromthe column at high salt were considered to represent frag-ments of TFIIIA that remained tightly bound to DNA. TheseTFIIIA fragments were fractionated by SDS-PAGE anddetected by immunoblotting with the anti-TFIIIA N-terminalantibody (Fig. 5B) or by Coomassie blue staining (Fig. 5C).This analysis revealed that the 280- and 220-amino-acidN-terminal fragments of TFIIIA remained bound to theintact 5S DNA ICR. In contrast, the major fragment retainedon the DEAE column by association with the AS' + 69fragment was the N-terminal 200-amino-acid fragment. Itshould be noted that a slightly greater yield of shorterproducts was observed in Fig. 5 than in Fig. 4 presumablybecause trypsin treatment is stopped less effectively with theDEAE minicolumn procedure. The experiments in Fig. 4and 5 analyzing the relatively well-understood interaction ofTFIIIA with 5S DNA establish methods to characterize theinteraction of TFIIIA with 5S RNA.The central zinc fingers of TFUIIA associate most tightly

with 5S RNA. Proteolytic footprinting experiments wereperformed to compare the products of trypsin treatment ofparticles assembled with either somatic- or oocyte-type SSRNA with the products obtained with the naturally occurring7S particle. Figure 6 shows the results of two separateexperiments to analyze the products of trypsin treatmenteither on native agarose gels (Fig. 6A) or by immunoblotting(Fig. 6B). Trypsin treatment of the naturally occurring 7Sparticle produces a single discrete subparticle (Fig. 6A, lanes2 and 3) that presumably contains nine- and seven-fingerfragments of TFIIIA, as detected by immunoblotting of aparallel preparation (Fig. 6B, lanes 6 and 7). Ribonucleopro-tein (RNP) particles reassembled by binding TFIIIA to eitheroocyte-type or somatic-type 5S RNA had the same gelmobility as did the naturally occurring 7S particle (Fig. 6A).However, trypsin treatment of the reassembled RNP parti-cles generated more complex mixtures, including particleswith increased gel mobility. Trypsin treatment of the RNPcontaining oocyte 5S RNA and TFIIIA produced a fragmentwith an apparent mobility the same as that of the trypticfragment of the naturally occurring 7S particle (comparelanes 6 and 7 with lanes 2 and 3 in Fig. 6A) as well as a morerapidly migrating particle. Immunoblotting of the TFIIIAdigestion products from a parallel preparation revealed frag-ments containing the N-terminal =280 residues (Fig. 6B,lanes 8 and 9). Trypsin treatment of the particles reassem-bled by binding TFIIIA to somatic 5S RNA reproduciblyproduced a slightly different pattern of products. This digestcontained a greater relative yield of the seven-finger frag-ment of TFIIIA (Fig. 6B, lanes 10 and 11). For bothreassembled RNP particles, immunoblotting with the N-ter-minal antiserum did not reveal specific proteolytic fragmentsthat might account for the particles with greater mobility onthe native gels (Fig. 6A, lanes 6 through 9). This finding isconsistent with the possibility that the N-terminal fingers ofTFIIIA were removed from these rapidly migrating particlesby trypsin digestion.An additional experiment was performed in an effort to

determine whether internal fragments of TFIIIA might re-

MOL. CELL. BIOL.

PROTEOLYTIC FOOTPRINTING OF TFIIIA 5155

A A7S 0-5S RN-A S-5S RPNA

1 9 3 4 3 6 7 8 9 10 111 2 3 4 5

-7S

-5S RNA

7S-

5S-

BA+ A+

i1 ~ C, n -7 Q C7 IQMf A { u-U^a o

1 2 3 4 5 6 7 8 9 1011

a=- IW Wbjm3

b- -- - - * _

FIG. 6. Proteolytic footprinting of TFIIIA complexed with 5SRNA. (A) Ethidium bromide-stained gel of 5S RNA-protein com-plexes. Lanes: 1, mobility of 7S particles isolated from immatureoocytes; 2 and 3, identical samples of 7S particles treated for 1 minwith 1 and 3.3 times the standard concentration of trypsin; 4 and 5,oocyte-type 5S RNA without and with TFIIIA; 6 and 7, productsobtained by treating the reassembled TFIIIA-oocyte 5S RNA com-

plexes with trypsin as in lanes 2 and 3; 8 through 11, identical tolanes 4 through 7 except that somatic-type 5S RNA was used. Themobilities of free 5S RNA and intact 7S particles are shown on theleft. (B) Immunoblot of proteolytic footprints. Lanes 4 and 5 showthe products of treatment of free TFIIIA with increasing concentra-tions of trypsin. Lanes 6 through 11 contain tryptic productsobtained with naturally occurring 7S particles (lanes 6 and 7),particles reassembled with oocyte-type 5S RNA (lanes 8 and 9), andparticles assembled with somatic 5S RNA (lanes 10 and 11).Samples in lanes 4, 6, 8, and 10 were treated with the standardconcentration of trypsin, while those in lanes 5, 7, 9, and 11 weretreated with a 3.3-fold greater quantity of trypsin as in panel A.Lanes 1 and 2 contain intact TFIIIA and a recombinant proteincontaining residues 1 to 220 of TFIIIA. Lane 3 contains the Asp-Nendoproteinase markers described for Fig. 2C. Letters on the leftindicate the mobilities of fragments of TFIIIA with 228 (a), 208 (b),194 to 184 (c), and 142 (d) N-terminal residues.

main associated with Xbs 5S RNA during chromatographyon DEAE-Sephacel (Fig. 7). Native gel electrophoresisconfirmed that trypsin treatment of RNP particles assembledwith somatic 5S RNA generated a product with a gelmobility greater than that of the trypsin-treated naturallyoccurring 7S particle (compare lanes 2 and 5 in Fig. 7A). Theproteins retained on DEAE-Sephacel by association with 5SRNA were analyzed in Fig. 7B and C. As expected, trypsintreatment of the naturally occurring 7S particle produced=220- and =280-amino acid fragments of TFIIIA retainingthe N-terminal epitope (Fig. 7B and C, lanes 3; compare withFig. 3A, lanes 4 and 5). Trypsin treatment of the reassembledparticle produced minor amounts of 220- and 200-amino-acidfragments of TFIIIA retaining the N-terminal epitope alongwith a more intensely staining band of approximately 17 kDathat does not contain the N-terminal epitope (Fig. 7B and C,lanes 4). Since this fragment did not react well with the

B1 2 3 4 L

A- _ -319

- g-194/109142

1 2 3 4 M

A-_ ~~~31

-20.4/19.7- 16.9

U - 14.4v8.1/6.9

FIG. 7. An internal fragment of TFIIIA remains associated with5S RNA after trypsin treatment of the reassembled TFIIIA-RNAcomplex. (A) Native agarose gel analysis. An ethidium bromide-stained agarose gel of TFIIIA-RNA complexes is shown. Lanes: 1and 2, 7S particle before and after treatment with trypsin for 1 mi;3, free Xbs 5S RNA; 4, mixture of free and TFIIIA-bound Xbs 5SRNA resulting from incubation of TFIIIA with a 1.5-fold excess of5S RNA; 5, trypsin products of the in vitro assembled TF1IIA-5SRNA complexes. (B) SDS-PAGE analysis of fragments detected byimmunoblotting. Naturally occurring 7S particles (lane 3) or reas-sembled 5S RNA-TFIIIA complexes (lane 4) were treated withtrypsin for 1 min and loaded onto a small DEAE-Sephacel column asdescnrbed in Materials and Methods. Proteins bound to the DEAEcolumn were eluted with 0.5 M NaCl and analyzed by SDS-PAGEand immunoblotting with the anti-N-terminal antiserum. Lanes 1and 2 show markers of intact TFIIIA and an amino-terminal220-residue seven-finger fragment of TFIIIA produced in E. coli;lane L contains the Asp-N fragment ladder described for Fig. 2.Sizes are indicated in amino acid residues. (C) SDS-PAGE analysisof fragments detected by Coomassie blue staining. Lanes 1 through4 are as described for panel B; lane M contains low-molecular-weight protein markers with the sizes (in kilodaltons) as indicated.

anti-N-terminal antiserum, it was presumed to be an internalfragment of TFIIIA that remains tightly bound to 5S RNA. Aparallel preparation of this tryptic fragment was subjected toN-terminal sequencing. The sequence obtained, FHNIKI-VYV (the hyphen represents a C residue lost during Edmandegradation), begins at residue 97 of TFIIIA. This RNA-binding fragment of TFIIIA most likely extends from thecleavage site after R-96 to the hypersensitive cleavage sitenear residue 220, including zinc fingers 4 through 7.

DISCUSSION

Proteolytic footprinting. The use of proteases as probes ofprotein structure is well established. Limited protease treat-ment has often been used to dissect functional or structuraldomains of proteins. The cleavage of immunoglobulins intoFc and Fab fragments (32) and the cleavage of DNA poly-

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5156 BOGENHAGEN

merase I to produce Klenow polymerase (22) are familiarexamples. Partial protease digestion has also been used tostudy the relationships between proteins (8) and to mapepitopes for monoclonal antibodies (36). However, in manycases, the pattern of partial proteolytic products can becomplex. Jue and Doolittle (21) have used an N-terminalchemical tag to simplify the analysis of chemical digests ofproteins. However, these chemical procedures are not com-patible with retention of biological activity for a nucleicacid-binding protein. In this study, I have developed animmunoblotting strategy to identify proteolytic fragmentsthat retain the N-terminal epitope. Since the N terminus isretained in any fragment that is identified by immunoblot-ting, the size of a fragment directly determines the newcarboxyl terminus generated by proteolysis. With appropri-ate markers (Fig. 2), the site of protease cleavage can beidentified within a few amino acids. In this study, I haverelied on a single N-terminal peptide antiserum. This isparticularly useful for the case of TFIIIA, since the Nterminus is adjacent to the zinc finger domains involved inbinding to nucleic acids. I have not used a C-terminal probeto analyze TFIIIA since the C-terminal domain is readilyremoved by proteases and is not involved in binding tonucleic acids (Fig. 3). However, for other proteins, an anti-serum directed against the C terminus or an N- or C-terminalepitope tag added by using recombinant DNA techniquesmight be useful (29). Lindsley and Wang (26) have used thissort of epitope label to study changes in protease accessibil-ity of topoisomerase II resulting from binding of ATP.

In this study, proteolytic footprinting was applied to theanalysis of the interaction of transcription factor TFIIIAwith both 5S DNA and 5S RNA. I have compared patterns ofproteolytic fragments of the free protein with those producedfrom protein complexed with 5S DNA or 5S RNA. Thestrategy for proteolytic footprinting was adapted from thatused for DNase I mapping. Very brief treatments with ratherhigh concentrations of protease were used to clip the mostaccessible sites in the protein. Comparison of protease-sensitive sites in free and bound protein can indicate regionsof the protein that may be protected from proteolysis or thatmay undergo a significant change in conformation uponbinding to nucleic acid. When this sort of proteolytic foot-printing procedure is used to compare patterns of cleavage infree and bound states of the protein, the method requiresthat all of the protein be complexed with nucleic acid. Theanalogous limitation applies to DNase I footprinting ofnucleic acids as well, since the inability to saturate a bindingsite in DNA with a specific binding protein provides only apartial footprint. TFIIIA provides an ideal subject for thisapproach. The free protein has an elongated conformation(reference 2, as measured in the presence of EDTA), so thatpotential sites for proteolysis are accessible. In addition, thepurified protein retains excellent nucleic acid binding activ-ity.Given the large number of potential cleavage sites in

TFIIIA for trypsin and chymotrypsin, it is not surprising thatit is often difficult to identify a single cleavage site respon-sible for generation of a particular product. Even in the freeprotein, some sites are cleaved by protease much morereadily than others. Both trypsin and chymotrypsin aremuch more likely to cleave in the non-zinc finger domainthan in the zinc fingers. Treatment of the free protein withEDTA to chelate the zinc greatly increases the probability ofcleavage within the zinc finger domains (2a). Thus, thecoordination of zinc with the apoprotein may wrap individ-ual zinc finger domains into more compact, protease-resis-

tant shapes. In all of the experiments reported in this paper,TFIIIA prepared and handled in the presence of zinc wasused.TFIIIA has distinct tight binding sites for 5S RNA and 5S

DNA. Binding of TFIIIA to either 5S RNA or 5S DNAproduces similar changes in the pattern of proteolysis (Fig.3). The enhancement of protease cleavage following finger 9of TFIIIA bound to 5S RNA or 5S DNA is consistent withthe possibility that all nine fingers are involved in binding toeither 5S RNA or 5S DNA, as suggested by Darby and Joho(9). In either RNA or DNA complexes, some zinc fingersbind more tightly than others. When TFIIIA binds to 5SDNA, the binding of the three N-terminal zinc fingers to theC-box element of the ICR can be considered a nucleationevent. This interaction brings the more C-terminal fingersinto proximity with the 5' elements of the ICR. Fingers 4through 9 of TFIIIA do not bind significantly to DNA in theabsence of fingers 1 through 3; intact TFIIIA does not bindwell to deletion mutants of TFIIIA lacking box C. Datapresented here suggest that fingers 4 through 7 of TFIIIAmay contain a similar tightly binding site for 5S RNA.

In the experiments whose results are shown in Fig. 6 and7, I have taken advantage of the observation that trypsinfootprinting has revealed subtle differences between natu-rally occurring 7S particles and particles reassembled bybinding TFIIIA to 5S RNA. Trypsin treatment of the reas-sembled particles produces complexes with greater mobilityin native gels that appear to contain a smaller fragment ofTFIIIA. Figure 7 shows that an internal fragment of TFIIIAremains tightly bound to 5S RNA in native gels and duringDEAE-Sephacel chromatography. This fragment was notobserved in the initial protease footprinting experiments(Fig. 6) because trypsin cleavage at R-96 removes fingers 1through 3, including the N-terminal epitope. The fragmenttightly bound to somatic 5S RNA extends from F-97 toapproximately R-218, including precisely fingers 4 through 7of TFIIIA. The simplest interpretation of these observationsis that the central fingers of TFIIIA represent a tightlybinding site for 5S RNA in the same sense that fingers 1through 3 represent a tightly binding site for 5S DNA.Following submission of this report, Clemens et al. (7)presented an analysis of the 5S RNA binding abilities of aseries of recombinant derivatives of TFIIIA showing thatfingers 4 through 7 were necessary and sufficient for tightbinding of TFIIIA to 5S RNA. This finding is in excellentagreement with the results observed with protease footprint-ing. Theunissen et al. (38) also concluded that the centralfingers of TFIIIA are most important for binding RNA,although their binding experiments with more complex re-combinant constructs failed to show a requirement for finger7.RNP particles reassembled with oocyte SS RNA resemble

naturally occurring 7S particles more closely than do parti-cles reassembled with somatic 5S RNA. Although these twoRNAs are considered to have similar structures (40), theydiffer at several sites, including a cluster of base changes inhelix III and loop B. The two RNAs have different mobilitiesin partially denaturing gels (39). Romaniuk et al. (33) havesuggested that a sequence difference at residue 79 mayincrease the stability of helix IV/V in oocyte 5S RNA. Thegreater apparent stability of the naturally occurring 7Sparticle might also reflect the effects of prolonged contactbetween the RNA and protein in vivo or the action ofunknown assembly factors in the oocyte. A comparison ofRNase footprints of intact and deleted derivatives of TFIIIAto oocyte, somatic, and hybrid SS RNA genes is in progress.

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PROTEOLYTIC FOOTPRINTING OF TFIIIA 5157

The problems encountered in trying to reconstitute 7Sparticles raise some questions concerning previous RNasefootprinting experiments to map contacts between 5S RNAand TFIIIA. In a comparison of the RNase footprints ofintact TFIIIA and the seven-finger 20-kDa tryptic fragment,Sands and Bogenhagen (34) suggested that slight changes inRNase cleavage in the tip of helix V of Xbs 5S RNA mightreflect interaction of this region with fingers 8 and 9 ofTFIIIA. Theunissen et al. (38) suggested that fingers 1through 3 of TFIIIA are directed toward the tip of helix V.However, sequences at the tip of helix V are clearly dispens-able for tight binding of TFIIIA to 5S RNA (4), and fingers 1through 3 do not contribute significantly to the bindingaffinity (7).

ACKNOWLEDGMENTSThis research was supported by a grant from the NIH (GM 33321)

and by fellowship support from the American Cancer Society andthe John Simon Guggenheim Foundation. The initial protease foot-printing experiments were performed while I was on sabbaticalleave in the laboratory of Aaron Klug at the Medical ResearchCentre, Cambridge, United Kingdom.

I thank Tom Fisher of the Center for Analysis and Synthesis ofMacromolecules at SUNY-Stony Brook for synthesis of peptidesand for protein sequencing. I also thank Aaron Klug, Paul Fisher,and Sid Strickland for comments on the manuscript.

REFERENCES1. Allen, G. 1989. Sequencing of proteins and peptides. Elsevier,

Amsterdam.2. Bieker, J. J., and R. G. Roeder. 1984. Physical properties and

DNA-binding stoichiometry of a 5S gene-specific transcriptionfactor. J. Biol. Chem. 259:6158-6164.

2a.Bogenhagen, D. F. Unpublished data.3. Bogenhagen, D. F. 1985. The intragenic control region of the

Xenopus 5S RNA gene contains two factor A binding domainsthat must be aligned properly for efficient transcription initia-tion. J. Biol. Chem. 260:6466-6471.

4. Bogenhagen, D. F., and M. S. Sands. 1992. Binding of TFIIIA toderivatives of 5S RNA containing sequence substitutions ordeletions defines a minimal TFIIIA binding site. Nucleic AcidsRes. 20:2639-2645.

5. Christensen, J. H., P. K. Hansen, 0. Lillelund, and H. C.Thogersen. 1991. Sequence-specific binding of the N-terminalthree-finger fragment of Xenopus transcription factor IILA to theinternal control region of a 5S RNA gene. FEBS Lett. 281:181-184.

6. Clemens, K. R., X. Liao, V. Wolf, P. E. Wright, and J. M.Gottesfeld. 1992. Definition of the binding sites of individual zincfingers in the transcription factor IIIA-5S RNA gene complex.Proc. Natl. Acad. Sci. USA 89:10822-10826.

7. Clemens, K. R., V. Wolf, S. J. McBryant, P. Zhang, X. IUno,P. E. Wright, and J. M. Gottesfeld. 1993. Molecular basis forspecific recognition of both RNA and DNA by a zinc fingerprotein. Science 260:530-533.

8. Cleveland, D. W. 1983. Peptide mapping in one dimension bylimited proteolysis of sodium dodecyl sulfate-solubilized pro-teins. Methods Enzymol. 96:222-229.

9. Darby, M. K., and K. E. Joho. 1992. Differential binding of zincfingers from Xenopus TFIIIA and p43 to 5S RNA and 5S RNAgenes. Mol. Cell. Biol. 12:3155-3164.

10. Del Rio, S., and D. R. Setzer. 1991. High yield purification ofactive transcription factor IIIA expressed in E. coli. NucleicAcids Res. 19:6197-6203.

11. Fairall, L., and D. Rhodes. 1992. A new approach to the analysisof DNase I footprinting data and its application to the TFIIIA/5SDNA complex. Nucleic Acids Res. 20:4727-4731.

12. Fruton, J. S. 1975. The specificity of proteinases toward proteinsubstrates, p. 33-50. In E. Reich, D. B. Rifkin, and E. Shaw(ed.), Proteases and biological control. Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y.

13. Galas, D. J., and A. Schmitz. 1978. DNAase footprinting: asimple method for the detection of protein-DNA binding spec-ificity. Nucleic Acids Res. 5:3157-3170.

14. Ginsberg, A. M., B. 0. King, and R. G. Roeder. 1984. XenopusSS RNA gene transcription factor, TFIIIA: characterization of acDNA clone and measurement of RNA levels throughout de-velopment. Cell 39:479-489.

15. Gralla, J. D. 1985. Rapid "footprinting" on supercoiled DNA.Proc. Natl. Acad. Sci. USA 82:3078-3081.

16. Hanas, J. S., D. F. Bogenhagen, and C. Wu. 1983. Cooperativemodel for the binding of Xenopus transcription factor A to the5S RNA gene. Proc. Natl. Acad. Sci. USA 80:2142-2145.

17. Hanas, J. S., A. L. Duke, and C. J. Gaskins. 1989. Conforma-tional states of Xenopus transcription factor IIIA. Biochemistry28:4083-4088.

18. Hayes, J., and T. D. Tullius. 1992. Structure of the TFIIIA-5SDNA complex. J. Mol. Biol. 227:407-417.

19. Hayes, J., T. D. Tullius, and A. P. Wolffe. 1989. A protein-protein interaction is essential for stable complex formation ona 5S RNA gene. J. Biol. Chem. 264:6009-6012.

20. Honda, B. M., and R. G. Roeder. 1980. Association of a5S genetranscription factor with SS RNA and altered levels of the factorduring cell differentiation. Cell 22:119-126.

21. Jue, R. A., and R. F. Doolittle. 1985. Determination of therelative positions of amino acids by partial specific cleavages ofend-labeled proteins. Biochemistry 24:162-170.

22. Klenow, H., and I. Henningsen. 1970. Selective elimination ofthe exonuclease activity of the deoxyribonucleic acid poly-merase from Escherichia coli B by limited proteolysis. Proc.Natl. Acad. Sci. USA 65:168-175.

23. Laemmli, U. K. 1970. Cleavage of structural proteins during theassembly of the head of bacteriophage T4. Nature (London)227:680-685.

24. Lerner, R., N. Green, H. Alexander, F.-T. Liu, J. G. Sutcliffe,and T. M. Shinnick. 1981. Chemically synthesized peptidespredicted from the nucleotide sequence of the hepatitis B virusgenome elicit antibodies reactive with the native envelopeprotein of Dane particles. Proc. Natl. Acad. Sci. USA 78:3403-3407.

25. Liao, X., K R. Clemens, L. Tennant, P. E. Wright, and J.Gottesfeld. 1992. Specific interaction of the first three zincfingers of TFIIIA with the internal control region of the Xeno-pus SS RNA gene. J. Mol. Biol. 223:857-871.

26. Lindsley, J. E., and J. C. Wang. 1991. Proteolysis patterns ofepitopically labeled yeast DNA topoisomerase II suggest anallosteric transition in the enzyme induced by ATP binding.Proc. Natl. Acad. Sci. USA 88:10485-10489.

27. McConkey, G. A., and D. F. Bogenhagen. 1988. TFIIIA bindswith equal affinity to somatic and major oocyte SSRNA genes.Genes Dev. 2:205-214.

28. Miller, J. R., E. M. Cartwright, G. G. Brownlee, N. V. Federoff,and D. D. Brown. 1978. The nucleotide sequence of oocyteSSDNA in Xenopus laevis. II. The GC-rich region. Cell 13:717-725.

29. Munro, S., and H. R. B. Pelham. 1984. Use of peptide tagging todetect proteins expressed from cloned genes: deletion mappingfunctional domains of Drosophila hsp 70. EMBO J. 3:3087-3093.

30. Pelham, H. R. B., and D. D. Brown. 1980. A specific transcrip-tion factor that can bind either the 5S RNA gene or SS RNA.Proc. Natl. Acad. Sci. USA 77:4170-4174.

31. Peterson, R. C., J. L. Doering, and D. D. Brown. 1980. Charac-terization of two Xenopus somatic 5S DNAs and one minoroocyte-specific 5S DNA. Cell 20:131-141.

32. Porter, R. R. 1973. Structural studies of immunoglobulins.Science 180:713-716.

33. Romaniuk, P. J., I. L. de Stevenson, C. Ehresmann, and B.Ehresmann. 1988. A comparison of the solution structures andconformational properties of the somatic and oocyte SS rRNAsof Xenopus laevis. Nucleic Acids. Res. 16:2295-2312.

34. Sands, M. S., and D. F. Bogenhagen. 1991. The carboxyterminalzinc fingers of TFIIIA interact with the tip of helix V of 5S RNAin the 7S ribonucleoprotein. Nucleic Acids Res. 19:1791-1796.

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35. Shang, Z., W. T. Windsor, Y.-D. Liao, and C.-W. Wu. 1988.Purification of Xenopus transcription factor IIIA and 5SRNAfrom 7S ribonucleoprotein particle by ammonium sulfate pre-cipitation. Anal. Biochem. 168:156-163.

36. Sheshberadaran, H., and L. G. Payne. 1989. Protein footprintingmethod for studying antigen-antibody interactions and epitopemapping. Methods Enzymol. 178:746-764.

37. Smith, D. R., I. J. Jackson, and D. D. Brown. 1984. Domains ofthe positive transcription factor specific for the Xenopus 5SRNA gene. Cell 37:645-652.

38. Theunissen, O., F. Fudt, U. Guddat, H. Mentzel, and T. Pieler.1991. RNA and DNA binding zinc fingers in Xenopus TFIIIA.

Cell 71:679-690.39. Wakefield, L., and J. B. Gurdon. 1983. Cytoplasmic regulation

of 5S RNA genes in nuclear-transplant embryos. EMBO J.2:1613-1619.

40. Westhof, E., P. Romby, P. J. Romaniuk, J.-P. Ebel, C. Ehres-mann, and B. Ehresmann. 1989. Computer modeling fromsolution data of spinach chloroplast and of Xenopus laevissomatic and oocyte 5S rRNAs. J. Mol. Biol. 207:417-431.

41. You, Q., and P. J. Romaniuk. 1990. The effects of disrupting 5SRNA helical structures on the binding of Xenopus transcriptionfactor IIIA. Nucleic Acids Res. 18:5055-5061.