Chemical probing of conformation in large RNA molecules

J. Mol. Riol. (1984)

180.

151-177

Chemical Probing of Conformation RNA Molecules Analysis of 16 S Ribosomal

in Large

RNA using Diethylpyrocarbonate

BARBARA J. VAN STOLK AND HARRY F. NOLLER ‘I’himann

Laboratories,

University

Santa Crux, CA 95064, (Received

of California Ly.R.A.

11 May 1984, and in revised form

12 July

1984)

Peattie & Gilbert (1980) have described an accurate and rapid gel method for assessing conformation of individual nucleotides in RNA, based on chemical modification of bases and aniline-induced strand scission. In order to extend this approach to analysis of large RNA molecules, we introduce the use of hybridization of modified RNA with DNA restriction fragments to generate RNA fragments of defined length. In principle, this permits chemical probing of conformation at any position of any RNA molecule for which a cloned DNA coding sequence is available. To illustrat,e the utility of this method, we use diethylpyrocarbonate to probe the reactivities of adenine residues in Escherichia coli 16 S rRNA under “native” (80 mkr-potassium cacodylate (pH 7.0), 20 mM-MgCl,. 300 mM-KCl) and “quasisecondary“ (80 mM-potassium cacodylate (pH 7*0), 1 mM-EDTA) conditions. This study shows that: (1) there is generally good agreement between diethylpyrocarbonate reactivities of adenine residues in naked 16 S rRNA and a secondary structure model based on comparative sequence analysis: of 309 adenine residues probed under native conditions, only four strongly reactive residues are found in helices in the model. (2) Candidates for possible tertiary interactions are identified as adenine residues that are unpaired in the model and unreactive toward diethylpyrocarbonate under native conditions but’ reactive under quasi-secondary conditions. (3) An unexpectedly stable structure has been identified in the region between positions 109 and 279, where many adenine residues remain unreactive even at 90°C in 80 mM-potassium cacodylate. 1 mM-EDTA. This may correspond to a structural “core” t,hat is important for early events in ribosome assembly.

1. Introduction (lonsiderable structure

progress has been made in recent years toward

of ribosomal

RNAs,

and their role in protein

the elucidation

synthesis

of the

(for reviews,

see

Rrimacombe et aZ., 1983; Woese et al., 1983; Noller, 1984). Nucleotide sequences for a great many large rRNAs from organisms representing a wide phylogenetic range have been determined; these structures can all be folded into secondary structures that share a common general base-pairing scheme. These secondary

152

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\‘AN

STOLK

ANI)

H.

It’. XOLLEK

structure models have been developed largely from comparative sequence analysis, but, are supported by a wide range of experimental evidence; furthermore, there is good agreement between the models developed independently by three research groups (Woese of nl.. 1983; Krimacombe et nl., 1983; Stiegler et al., 1981). These findings have served t,o focus our attention on new questions. Do the static models accurately describe the secondary structure of rRNA in a functioning ribosome, or do alt’ernative (“switch”) structures exist for cert,ain structural elements, indicative of possible machine-like movements of the RSA during translation? In the absence of X-ray crystallographic analysis. can we identify element,s of t,rrtiarg structure! And, what) are the precise sites of interaction of t)he ribosomal proteins and various functional ligands with rRNA? (‘hemical modification provides one of the most sensitive and specific approaches to these problems. This method is generally preferable to enzymat’ic (nuclease) probing, since cleavage of the R#NA backbone can be avoided, and because the small size of most chemical probes allows their access t,o more of the macromolecular structure. Studies on tRXA have shown that several chemical reagents are extremely accurate and reliable probes of secondary and tertiary struct’ure (Rhodes, 1975; Peattie & Gilbert’, 1980: de Bruijn & Klug, 1983). I’nfortunately. it is difficult to apply t’hese methods to large molecules such as t’he ribosomal KXAs. Fingerprint’ or “diagonal” methods (e.g. see Noller, 1974) suffer from a lack of resolution, and from the difficult’y of placing small oligonucleotides in a long sequence. Gel methods are limibed by their resolving power tjo regions of about, 200 nucleotides in length. Moreover. it. is generally difficult to obtain specific fragments of large Rh’A molecules, owing mainly to the unavailability of ribonucleases with specificities comparable to t,hose of the DNA restriction endonucleases. In this paper. we describe a method for the isolation of specific RNA fragments of manageable size by hybridization to restriction fragments of the corresponding RNA gene. The method exploits t’he resistance of RNA in DNA-RNU’A hybrids to digestion by single-starand-specific RNase. As the RNA fragments retained in t,he hybrids are readily end-labeled, the s&es of modification can be identified easily by aniline-induced chain cleavage (Peattie, 1979). Experiment’s reported herein describe the preliminary application of this approach using diet’hylpyrocarbonate as a chemical probe of the conformation of 16 S rRNA from Escherichia co&.

2. Materials (a) O~wrall

and Methods sfrcxtegy

DNA containing the rRKA genes is restricted, and hybridized with chemically modified 16 S rRNA under conditions favorable to formation of RSA-DNA hybrids (Thomas et al., 1976; Casey 8: Davidson, 1977). Treatment with RXase T, trims away non-hybridized RNA, which is removed by filtration through a column of Sephadex beads. RNA in the hybrids remains intact during this treatment and is end-labeled either at the 3’ end with [32P]pCp or at the 5’ end with [y-“P]ATP. The labeled hybrids are then separated by electrophoresis on a non-denaturing polyacrylamide gel. Following elution, the purified hybrids are heat-denatured, so that the RNA fragments can be separated subsequently from the DNA components and repurified by electrophoresis through a denaturing

(‘ONFORMATIONAL

PRORING

OF

I6

S rRSA

I.3

polvacrylamide gel. The purified RNA fragments are subjected to aniline-induced chain n&&ion. allowing the sites of modification to be identified by the mobilities of the resulting end-labeled fragments after electrophoresis through a denaturing polyacrylamide gel. (b) Enzymes C’alf intestinal alkaline phosphatase (Sigma) was dialyzed at 4°C against 20 mM-Tria H(‘I (pH 8.6). 0.01 mM-ZnCl, and stored at -20°C at 900 to 1000 units/ml. RNA ligase was purchased from PL Biochemicals, and restriction enzymes were purchased from either NW England Biolabs or Bethesda Research Labs. (c) Buffers

Buffers used for modification of RNA were CMK buffer (80 mw-potassium cacodylate (pH 7.0). 20 mM-MgCl,. 300 mM-KCl) and CEDTA buffer (80 mM-potassium cacodylate (pH 7.0). I mM-EDTA). Other buffers used for examination of cytidine modification were (‘M buffer (80 rnM-potassium cacodylat,e (pH 7.0). 20 m&I-MgCl,) and CK buffer (80 mM-potassium cacodylat,e (pH 7.0). 300 mw-KCl). Hybridization buffer was 89% (v/v) deionized formamide, 0.44 M-NaCl, 19 rnM-NaHepes (pH 7.0). Ligation buffer was -50 mnr-NaHeprs (pH 7.5), 3.3 mM-dithiothreitol, 15 mM-Mg(‘l,, lo?;, (v/v) dimethglsulfoxide. 0.01 mg bovine serum albumin/ml. Elution buffer was 0.3 M-sodium acetate. 0.2°, (IV/V) sodium dodecyl sulfate. 0.05 mg carrier tRN\;A,‘ml. TBE is 89 mw’rris-boratr. .5 mwEDT.4 (pH 8.3). (d) Plasmid

construction

pJS53. a derivative of pKK3535. was constructed by J. Normanly in this laboratory to increase the yields of rDNA-containing plasmid by removing the strong ribosomal RNA promoter. pKK3535 (Brosius et al.. 1981a), was digested with BamHI and BclI, followed by religation. The resulting plasmid contains all of the 16 S and 23 S rRXA genes. except for the first 14 nucleot,ides of 16 S rRN4. (e) D,\‘A

restriction

For each hybridization experiment, 35 to 40 pg of pJN53, prepared as described (Brosius ut al.. 1978). was restricted according to the conditions recommended by the supplier fol each endonuclease. The digest was then brought to a volume of 300~1 and adjusted to 0.3 M-sodium acetate and 0.5% SDS?. Protein was removed by 3 extractions, each with an equal volume of water-saturated phenol, followed by 3 or 4 extractions with diethyl ether (1 ml each). The DNA was then precipitated with 1 ml of 95”/0 ethanol. washed with 80?() t+hanol. and vacuum dried. (f) RNA

modijcation

Native reaction: 15 pg of 16 S rRNA was preincubated in 200 ~1 of CMK buffer at 42°C for 1 h and cooled on ice: 5 ~1 of diethylpyrocarbonate (Eastman) was added and the solution was incubated at 37°C for 30 min with occasional mixing. Sodium acetate (200 ~1. I.5 M) was added and the modified RNA was precipitated w-ith 1 ml of 95% ethanol. Pellets were resuspended in 200 ~1 of 0.3 M-sodium acetate, reprecipitated with 800 ~1 of 959, ethanol. washed m 8Oqb ethanol, and vacuum dried. The RNA can be hybridized at this point. Quasi-secondary reaction: the procedure was the same as for the native reaction. with the exception t)hat 16 S rRNA was preincubated and modified in 200 ~1 of CEDTA bufl’er. Denaturing reaction: 15 pg of 16 S rRNil was preincubated in CEDTA buffer at 90°C fol t .1bbreviations

used:

SDS,

sodium

dodecyl

sulfate;

Et,pyCO,

diethplpyrocsrbonnte.

154

1J. *J. VAN

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AND

H.

F. NOLLEK.

2 min: 5 ~1 of Et,pyC was added to the hot solution. which was then incubated at 90°C for an additional 12 min: 200 ~1 of cold 1.5 M-sodium aretate was added and the RNA recovered as described above. (CJ) Hybridization

and

RXase

7’,

digestion

The hybridization conditions are similar to those optimized for generating R-loops (Thomas et al., 1976; Casey & Davidson, 1977). RNA and DNA were prepared as described using 10 to 15 pg of RNA and 35 to 40 pg of DNA for each hybridization experiment. The dried DNA pellets were redissolved in 10 ~1 of hybridization buffer and incubated at 67°C for 10 t,o 15 min. This DNA solution was t,hen added to the dried 15 pg RNA pellet while hot. and the solution was incubated at 50°C for 30 min. Then 290 ~1 of cold 0.3 M-sodium acetate, 20 mnz-Tris . HCl (pH 7.7 to 7.9) was added and mixed well (it is important that the hybridizabion mixture is cooled and diluted quickly). Immediately following the hybridization step, 2 ~1 of 2.5 mg RNase T,/ml (5 pg/sample of 10 to 15 pg of RNA) was added and the solution incubated at 37°C for 30 min. The mixture was then chilled on ice, and SDS added to 05O/;. Protein was removed by 2 extractions. each with an equal volume of water-saturated phenol. followed by 3 extractions with diethyl ether (1 ml each). Hybrids were precipitated with 1 ml of 9596 ethanol. washed with SOo,b ethanol. and vacuum dried. (h) Phosphatase

reaction

The hybrid pellets were resuspended in 20 ~1 of 20 rn>f-Tris . HCI (pH 8.6): 1 ~1 (approx. 1 unit) of calf intestinal phosphatase was added to each solution. which was then gently mixed by hand and incubated at 37°C for 30 min. Another 1 ~1 of phosphatase solution was added and the solution was incubated at 37°C for another 30 min. The volume was brought to 300 ~1 with 0.3 M-sodium acetate, 0.5% SDS and phosphatase was removed by 2 extractions with phenol and 1 with chloroform. Hybrids were precipitated with 1 ml of 9504 ethanol and vacuum dried. (i) Column

purijcation

Sephadex G-50 (fine) was swollen in 0.3 M-sodium acetate by incubation at 67°C for 2 h. and the slurry poured into a 1 ml disposable pipette. The column was washed with about 2 vol. 0.3 M-sodium acetate before loading the samples. The hybrid samples, prepared and treated with phosphatase as described above, were redissolved in 40~1 of 20% (w/v) sucrose, 0.3 M-sodium acetate and eluted through the column in 0.3 M-sodium acetate. The void volume was discarded and the fraction containing the hybrids was collected. Nucleic acids in the collected volume were precipitated with 1 ml of 95% ethanol, washed with XOyO ethanol, and vacuum dried. (j) 3’ End-labeling

Following column purification, the hybrids were labeled with [32P]pCp at the 3’ end of the RNA (Bruce & Uhlenbeck, 1978). Each reaction contained 9 ~1 of ligation buffer, 1 ~1 of 0.5 mg ATP/ml, 0.5 to 1.0 ~1 of RNA ligase and about 30 $i of [32P]pCp. The solution was incubated on ice for at least 6 h. Alternatively, one can use 5’ end-labeling using [Y-~‘P]ATP and polynucleotide kinase; in this case, the phosphatase step is omitted, giving preferential labeling of the RNA, which contains a 5’-OH, over the DNA ends, which are 5’-phosphorylated. In this study, we use 3’-labeling exclusively. (k)

Puri&ation

of hybrids

and

RNA

fragrr~ents

Following ligation, 10 ~1 of 0.1 x TBE, 0.4% SDS, 50% (v/v) glycerol, 0.05% (w/v) xylene cyan01 and bromophenol blue were added and the samples vortexed vigorously.

(‘ONFORMATIONAL

PROBING

OF

I *xl

16 R rRNA

They were then loaded directly onto an Sq/, (w/v) polyacrylamide (1 : 30 (w/w) aqlamide/bis-acrylamide)/TBE gel (1.5 mm thick) and electrophoresed until the bromophenolblue dye marker had migrated 2/3 the length of the gel. Hybrid bands were visualized by autoradiography (usually a 30 to 40 min exposure on Kodak NS film), csut out. and eluted into 350 ~1 of elution buffer with vigorous shaking at room temperature overnight. Hybrids were precipitated with 1 ml of 95”i, ethanol, washed with 800$, ethanol. and vacuum dried. Pellrts from the previous step were redissolved in 10 ~1 of 0.1 x TBE, 8 M-urea loading bufYer. heated to 90°C for 3 min to melt the hybrids. and quickly chilled. They were t,hen rlectrophoresed through an W?, polyacrylamide (1 : 30 (n/w) ac,r?;lamide/bis-acr?Ilatnidr) X sf-urea/TBE gel (1.5 mm thick). which was run as hot as possible to prevent hybrids reforming. Bands were visualized by autoradiography (usually 2 to 4 h exposure on Kodak NS film). cut out, and eluted into 3.50~1 of elution buffer at 4°C overnight with vigorous shaking. The RNA was precipitated with 1 ml of 95O’b ethanol. washed wit,h EW,, csl-hanoI. and va(zuum dried. (I) Identijcation

of modijed

sites

Modified R?U’A was hybridized, end-labeled, and fragments were purified by gel (Jlectrophoresis through 2 gels (section (k), above). Following elution from the second gel. the purified RNA fragments were treated with aniline as described (Peattie. 1979) and c+ctrophoresed through 100/b polyacrylamide (1 : 30 (w/w) acrylamide/bis-acrylamide)/8 Murea/TBE gels (0.5 mm thick). Bands were visualized by autoradiography (usually 2 t,o 20 (lays exposure on Kodak XAR-5 film with an intensifying screen). Protected adenine rcGlues were identified by comparison with a reference ladder of adenine residues produced II~ modificwtion under denaturing conditions,

3. Results (a) Preparation

of speci$c

RXA

fragments

In these experiments, plasmid pJN53 DNA is restricted and hybridized with E. coli 16 S rRNA

without

prior

purification

of individual

restriction

fragments.

Positions of relevant restriction sites and the corresponding RNA fragments generated are shown in Figure 1. Following hybridization, non-hybridized RNA is trimmed away with RNase T,, and t,he RNA moiety in the hybrids is selectively 3’ end-labeled using RNA ligase.

AIUI HhUI

Mb*= ?l HpoII

tHtItJt -_-_-----_‘y

C t

c

tDtE+ I I

B

-------AA

t

tot--

B

t

tt ----.-L---J ttt --

---

1

t --

t

t

Fra. 1, Schematic reprrsrntation of E. coli I6 H rRSA. and restriction fragments from t,he corresponding gene from the rm B transcriptional unit (Brosius it al.. IC%la,b). Restriction sites are denoted by arrows. Thick lines show the extent of the RNA structure that was analyzed following hybridization with the respective restriction fragments; broken lines show the portion of the RSA fragment that was not resolved. Fragments denoted by letters are presented in Figs 2 and 3. I’nderlines connecting 2 restriction fragment,s indicat,e tandem hybrids (discussed in the text).

156

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AND

H.

F.

NOLLER

Figure 2 shows an autoradiograph of hybrid fragments thus obtained using unmodified 16 S rRNA and DNA fragments from AZuI and HhaT-restricted pJN53, after polyacrylamide gel electrophoresis. The strong hybrid bands correspond well to the predicted DNA restriction pattern within the rRNA coding sequence (Fig. 2, ALuI, bands B. C, D, E. H. I and J; HhnI, bands A, B, C and D; cf. Fig. 1). Some unanticipitated weaker bands (Fig. 2. AZuT. bands A, F and G) were produced as a result of rRNA hybridizing simultaneously to two adjacent restriction fragments. Such “tandem” hybrids have been observed in some cases using other restrict*ion fragments and may also account for the very long fragments near the top of the gel. Figure 3(a) shows the isolation of RNA fragments obtained by denaturation of hybrids of unmodified 16 S rRNA and HhaI fragments C and D (cf. Fig. 1). In order to verify the location of each fragment in the 16 S rRNA sequence and to test for homogeneity at’ the labeled ends of the RNA, we subjected them to partial RNase T, digestion (Donis-Keller et al., 1977), as shown in Figure 3(b). The band positions correspond precisely t,o the location of guanine residues in the regions of I6 S rRNA encoded by restriction fragment-s C and D (Brosius et al., 1978). The fact that each band is a singlet demonstrates homogeneity at the labeled end; furthermore, RNase T, sensitivity confirms that separation of RNA from DNA was achieved. (b) Dejinition

of conditions

used for structural

probing

As an example of the application of this method, we describe the use of diethylpyrocarbonate to probe the conformation of naked E. coli 16 S rRNA. Et,pyCO reacts with adenine residues only when they are unstacked and free from interactions at N7 (Peattie & Gilbert, 1980). To help discriminate between protection of adenine residues as a consequence of secondary, as opposed to tertiary, structure, we monitor their reactivities under three sets of conditions, defined as follows: (1) Native. The 16 S rRNA conformation most generally accepted to approximate the native structure in the absence of ribosomal proteins is generated by heating the RNA under ionic condit,ions used for reconstitution in vitro (Traub &, Nomura. 1968). Here, we define native structure to be that’ resulting from preincubation in 80 mM-potassium cacodylate (pH 7.0), 20 mM-MgCl,, 300 mM-KC1 at 42°C for one hour. Modification is then performed at’ 37°C. (2) Quasi-secondary. In their studies on tRNA solution conformation, Peattie & Gilbert (1980) sought to disrupt tert’iary structure while maintaining secondary structure. based on the generally greater dependence of tertiary structure on Mg2 + concentration (Stein & Crothers, 1976; Crothers. 1979). Thus, following their approach, we define quasi-secondary structure as that generated by preincubation of 16 S rRNA in 80 mM-potassium cacodylate (pH 7-O), 1 mM-EDTA at 42°C for one hour, followed by modification at 37°C. (3) Denatured. Incubation of 16 S rRNA in 80 mM-potassium cacodylate (pH 7.0), 1 mM-EDTA at 90°C and modificat’ion at this temperature defines OUI denaturing condition.

(‘OXFORMATIONAL

PROBING A/U1

OF

16 S rRNA

l.i7

HhaI -or,

ABC-

DE-

-B -C

F-D xc G-

J-

FIG. 2. Isolation of RNA-DNA hybrids containing 3’-[32P]pCp-1abeled 16 S rRNA fragments and DNA fragments from Ah1 or HhuI restriction digests of pJN53 DNA. Hybrids were prepared as described in Materials and Methods, electrophoresed on an 8% polyacrylamide gel, and visualized by autoradiography. Hybrid bands are designated according to Fig. 1. Undesignated bands are not observed reproducibly and probably represent tandem hybrids. as described in the text. xc shows the position of the xylene ryanol marker and ori denotes the top of the gel.

158

H. J.

VAN

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BND

H.

F.

NOLLER

(b)

(a)

G474-

G524-

G542-

-%222 G558-

FIG. 3. Isolation of RNA fragments eluted, denatured, and run on an 8% Methods. The xylene cyan01 (xc) marker T, partial digests of RNA bands C and of specific guanine residue8 in the 16 indicated.

from hybrids (a) Hybrid bands Hhul C and D (Fig, 2) were denaturing polyacrylamide gel as described in Material8 and dye runs faster than fragment D and is not shown. (b) RNase D resolved on a 19 “/o denaturing polyacrylamide gel. Position8 8 rRNA sequence (BrOSiU8 et al., 1978) and xc marker are

(‘ONFORMATIONAL

PROBING

OF

16 S rRNA

I .i!l

Intact rRNA is modified under one of the above conditions. hybridized, treat’ed with RNase T,, and the resulting fragments end-labeled and purified by gel electrophoresis (see Materials and Methods). Treatment of the purified RNA fragments with aniline produces chain scission at the sites of modification. allowing their identification by gel electrophoresis and autoradiography (Peatt,ie k Gilbert, 1980). (c) The hybridization

procedure has no signijcant mod@cation pattern

e$f’ect on the

Because certain base modifications interfere wit,h formation of Watson-Crick base-pairs, it is critical to know whether or not the hybridization step selects against molecules whose basesare modified at certain positions. A priori, we did not anticipate t’hat’ this would be a serious problem, since only about 1% of the bases in a given molecule are modified under these conditions: this should not disrupt’ DNA-RNA interaction to the extent that the RNA chain would be cleaved within the hybridized region by treatment with RNase T,. To control for possible perturbations of the modification pattern introduced by t,he hybridization step, we modified the same region of the 16 8 rRNA before or after hybridization, and compared the results (Fig. 4). One sample of 16 S rRNA was modified with Et,pyCO under denaturing conditions and then hybridized to AU-rest,ricted pJN53 DNA (Fig. 4(a), lane 1); another sample of unmodified 16 S rRNa was first hybridized to AZ&-restricted pJN53 DNA (Fig. 4(a), lane 2) and then the purified RNA fragment C was modified with Et,pyCO under denaturing caonditions. The two samples of purified fragment C. one modified before hybridization. and the other modified after hybridization, were treat)ed with aniline and resolved on a denaturing polyacrylamide gel (Fig. 4(b)). No significant differences in the respective banding patterns are apparent. We conclude tha’t the hybridization procedure does not’ interfere with identification of sites of moditication in the RNA, nor with the relative intensities of t,he resulting bands. (d) Modijkation

of cytidine

by diethylpyrocarbonate

Previous st’udies using Et,pyCO as an RNA sequencing reagent (Peattie, 1979) or as a conformational probe (Peattie & Gilbert, 1980; Douthwait’e et al.. 1983a,b) have reported modification leading to aniline-induced chain scission only at adenine and guanine residues. During the course of these studies, we have reproducibly observed the occurrence of bands that do not correspond tjo the position of adenine or guanine residues. We observe 65 such bands, all of which caorrespond to cytosine residues. These do not resemble “band compression” artifacts. because they are quite frequent. are independent’ of the running t,emperature of the gel, and always show the correct spacing for aniline-induced st’rand scission at the position of cytosine residues in the 16 S rRNA sequence. The appearance of cytosine bands is dependent on experimental condit,ions (Fig. 5). Modification of I6 S rRNA with Et,pyCO either in 80 mw-potassium cacodylate (pH 7.0). 20 mM-MgCl,, 300 m&r-KC1 (Fig. 5. XAT lane) or in

(b)

(a)

-A563

-A574

-A563

-A596

-A602

xc -G

-407 -ho6 -A609

-H -I

FIG. 4. Comparison of modification patterns hybridization. (a) Hybrids containing DNA from Et,pyCO (lane 1) or unmodified 16 S rRNA (lane bands are marked according to the designations polyacrylamide gel of purified RNA from band C in lane 2 and aniline-induced chain scission. Lane RNA modified before hybridization. Positions of

obtained from RXA modified before or after AluI-restricted pJN.53 and 16 S rRNA modified with 2). separated on an go’/(, polvacrylamide gel. Hybrid in Fig. 1. (b) Electrophoret”ic separation on a lO’$; in (a), following modification with Et,pyCO of RNA 1 is RNA modified after hybridization, and lane 2 is specific adenine residues are indicated.

(‘ONFORMATIONAL C

DEN

PROBING SEC

NAT

OF CM

I61

16 S rRSA CK

J

-%18 A520A523-

-G524 G527-

FIG. 5. Autoradiograph of a lO”,c polyacrylamide gel showing Et,pyC’O reactivity of nucleotides in 16 S r-RN.4 between positions 408 and 527 (cf. Fig. 1, fragment HhnI C). Conditions of modification were ax described in Materials and Methods. NAT, modified under native conditions; SEC, quasisecondary conditions; DEN, denatured conditions: C. aniline-only control: CM, 80 mn-potassium cncodylate (pH 7.0). 20 mM-MgCl,; CK, 80 mr+r-potassium carodylate (pH i@), 300 mi+-KCl. Positions of specific residues are indicated. Reactive cytosine residues not indicated (NAT and CK lanes) are (‘422, (‘436. C440. C522 and C526. Rrackets show the extent of hrlicrs in the secondary structure model (Fig. 9; Worse rf /I/.. 1983).

162

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80 mM-potassium cacodylate (pH 7*0), 300 mM-KC1 (Fig. 5, CK lane) produces these bands, while modification in 80 mar-potassium cacodylate (pH 7*0), 1 mM-EDTA at 37°C or 90°C (Fig. 5, SEC and DEN lanes, respectively), or in 80 m&r-potassium cacodylate (pH 7*0), 20 mM-MgCl, (Fig. 5, CM lane), does not. (See, for example, bands corresponding to C469 and C507 in Fig. 5.) Cytidine bands are not found in the aniline-only control. We conclude that certain cytidine residues can be modified by Et,pyCO in a reaction that is dependent on the concentration of monovalent ions. We also notice a pronounced enhancement of modification of certain guanine residues in the low salt/Mg 2+ buffer (80 mxl-potassium cacodylate (pH 7*0), 20 mM-MgCl,). (See, for example, G481 and G524; Fig. 5, CM lane.) Although it is not clear why salt influences chemical reactivity of RNA bases, this result does demonstrate the need to interpret band intensities cautiously: a change in ionic conditions may alter chemical reactivity as well as accessibility of bases. (e) Detailed conformational

analysis

of speci$c sections of 16 S rRNA

Results from probing experiments on 16 S rRNA under native and quasisecondary conditions are catalogued in Table 1, along with the number of times each adenine residue was examined in separate experiments. Those results obtained from only a single experiment should be considered as preliminary. To provide examples of the kinds of results obtained in these studies, we consider in detail the chemical probing of four specific regions of E. coli 16 S rRNA.

TABLE

1

Reactivity of adenine residues in E. coli 16 S rRNA toward diethylpyrocarbonute under native and quasi-secondary conditions Modification

Position number

Native (CMK 37°C)

A44 A50 A51 A53 A55 A59 A60 A65 A66 A71 A72 A74 A77 A78 A80 A98

++ ++ + ++ ++ -

(1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (2)

conditions Quasisecondary (CEDTA, 37°C) nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd

Modification

Position number

Al01 A109 All6 A119 A120 Al29 A130 A131 4139 Al43 Al49 A151 A 152 Al55 Al60 A161

Native (CMK 37°C)

-

(2)

-

(3) (3) (3) (3) (3) (3) (3) (3) (3) (3) (3) (3) (3)

(2)

(2)

conditions secondary (CEDTA, 37°C) nd nd nd nd nd nd nd nd nd ILd(l) (1) (1) (1) ++ (1) (1)

CONFORMATIONAL

PROBING TABLE

Modification Native (CMK. 37°C) + + + +

(3) (3) (4) (4) (4) (4) (4) (4) (4) (4) (4) (4) (4) (4) (4) (4) (4) (4) (4) (4) (4) (4) (4) (4) (4) (4) (4) (4) (4) (3) (3) (3) (3) (3)

-

(3) (3) (3) (3) (3) (3) (3) (3) (3) (3) (3) (3) (3) (3)

+ +

(4) (4) (4) (-5)

conditions Quasisecondary (CEDTA, 37°C)

++ ++ ++ ++ ++ ++ ++ + + + + + + + + + + ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ +

(1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1)

OF

163

16 S rRNA

1 (continued) Modification

Position number

A482 A487 A493 A495 A496 A498 Al99 A.502 A509

A510 A520 A523 A532 A533 A535 A.539 A546 A547 A553 A.554 A.559 A560 A563 A572 A573 A574 Li579 A583 A59.5 A596 A600 Ad602 A607 A608 A609

A1621 A622 A629 A630 A635 A640 A642 A648 A649 A663 -1665 A673 A675 A676 A681 A687 A694

Native (CMK. -

conditions Quasisecondary (CEDTA.

37°C) + +

37°C) (5) (5) (f,) (5) (5) (5) (5) (5) (5) (5) (5)

(6) (7)

+ + -

(7) (7) (‘i) (7) (7) m (7) (7) (7) + + (5) + ~ -

(5) (4) (4) (3) (3) (3) (3) (3)

(3) + + (3) + (3) (2) + + (2) + + (2) (2) (2) (2) (2)

nd ++ ~ ++ ++ + +

(1) (1) (1) (1) (1) (1) (1) (1) 0) (3)

+ + + + ++ ++ ++ ++ ++ + + + +

(1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (2) (3)

+ + + + + + +

(3) (3) (3) (3)

+

(3) (3) (3) (3) (3) (3) (3) (2) (2) (2)

+ + + + + + + +

+ + + + + + + + + +

(2) (3 + + (2) (2) (2) + + (2) + + (2) + (1) ++ (1) ++ (1) (1) (1) (1) (1) (1)

nd nd nd nd nd lid nd nd nd nd

164

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Modification

Position number A695 A696 A702 8704 A706 B712 A715 A716 A718 A728 A729 A743 A746 A747 A749 A753 A766 A767 A768 A777 A780 $781 .478&z A784 A787 A790 A792 A794 A802 A807 A814 A815 A816 9819 A825 A831 A845 A878 A889 A892 A900 A901 A906 A907 A908 A909 A913 A914 A915 A918 A919 A923

Native (CMK 37°C) + + (3) + + (3) + + (3) + (3) (3) (3) + + (3) + + (3) + (3) + + (3) + + (3) + + (3) + + (3) + (3) (3) (3) (1) (1) (1) (1) (1) (1) (1) (1) (1) + (1) (1) (1) (1) (1) (1) (1) (1) + (1) (1) (1) ++ (1) (1) (3) (3) + (4) + (4) + + (5) + + (5) + + (5) (5) + + (5) + + (5) + + (5) + +

(5)

-

(5) (4)

conditions Quasisecondary (CEDTA. 37°C) nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ + + ++ ++ + +

(1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1)

‘Ld(l) + + (1) + (1) + (1) ++ (1) ++ (1) ++ (1) ++ (1) ++ (1) ++ (1) ++ (1) ++ (1) + + (1) (1)

AND 1

H.

F.

NOLLER

(continued) Modification

Position number A935 A937 A938 A946 A949 A958 A959 A964 A968 A969 A974 A975 A977 A978 A983 A994 A996 Al000 Al004 Al005 A1012 Al014 Al016 Al019 A1021 Al022 A1035 A1036 Al042 Al044 Al046 Al065 Al067 All17 All30 All45 Al 146 A1150 A 1 15 1 All52 All55 All57 All63 Al 167 All69 All70 All71 All76 A1179 A 1180 A1188 $1191

Native (CMK, 37°C) -

(4) (5) (5) (5)

+ +

(5) (5)

+ + + + + + nd + + + -

(5) (5) (5) (5) (5) (5) (5) (5) (4) (4) (4) (4) (4) C-1) (4) (4) (4) (4) (4) (4) (3) (3) (2) (2) (2) (1, (3) (5) (5) (5) (5)

-

(5)

-

(5) (5)

-

(5)

-

(5) + + (6) + (‘5) + (6) (6) (6) (6) (6) (6) (6)

conditions Quasisecondary (CEDTA, 37°C) nd ++ ++ ++ ++ ++ ++ + + + + + + ++ + + + + + + ++ ++ ++ ++ ++ ++ + + ++ + + + + + + + + + ++ ++ ++ ++ ++ + + + + + + + + + + + -

(1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (2) (2) (2) (2) (-4 (2) (2) (2) (2)

(‘ONFORMATIONAL

PROBING TABLE

Modification

Position number Al 196 Al 197 A1201 A1204 41213 Al216 A1219 A I225 Al227 Al 229 Al”3k Al 238 Al239 Al246 AI248 Al “50 Al251 Al252 Al254 Al%56 Al 257 Al261 Al%69 Al271 Al274 Al275 Al280 Al285 Al287 Al288 Al289 Al299 A 1306 Al311 A1318 A1319

Native (CMK, 37”(!) + + (6) (6) (6) (‘3) (‘3) (6) (6) (6) (‘3 + (‘3) (5) L

I;;

+ + + + + +

(b (1) (1) (1) (1) (1) (2) (2) (2) (2) (‘4 (a (2)

(2) (2) (3) (3) (3) (3) (3) (3) (4) (4)

conditions Quasisecondary (CEDTA, 37°C)

+ + (2) + + + + + + + + + + + ++

++ ++ + ++ + + ++ ++

(2) (2) (2) (2) c4 (4 (2) (t’)

(2) (4 (2) (2) (1) (2) (2) (2) (2) (2) (2) (2) (2) (2) (2) (2) (2) (2)

+ + (2) + + (2)

++ ++ + ++ +

(2) (2) (1) (1) iij (1) (1)

OF

16 S rRN.4

I 65

1 (continued) Modification

Position number Al324 Al329 Al332 Al333 91339 A1340 Al346 Al349 A1350 Al357 A1360 Al362 Al363 Al368 Al374 Al375 A137i Al394 81396 Al398 A1408 A1410 A1413 $1418 Al428 Al429 x1430 A1431 Al433 Al434 Al437 A1441 81446 Al447 Al456

Native (CMK. 37°C)

-

(4)

+ + -

(4) (4) (4) (4) (4) (4) (4) (4) (4) + + (4) + + (4) (4) (4) (4) (4) (4) ++ (1) (1) ++ (1) (1) (1) (1) PI (2) (a r4 (2) (a (2) (2)

++ + -

(2) (1) (1) (1)

conditions Quasisecondary (CEDTA. 37Y’)

++ ++ ++ -if ++ ++ ++ ++ ++ ++ ++ ++ ++

(1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1)

nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd

Positions refer to the sequence of E. coli 16 S rRNA from the rm B operon (Brosius ef al.. 1978,198 In). + + , Strongly reactive to Et,pyCO under the specified condition; + , weakly reactive: ~~. unreactive; nd, not determined. Conditions of modification were as described in Materials and Methods. Numbers in parentheses indicate the number of times each base was independently probed

(i)

The 408-527 region

Chemical probing of the 5’ domain shows this region of the 16 S rRNA to be highly structured. In the experiment displayed in Figure 5, for example, only two adenine residues (A411 and A412) show strong reactivity toward Et,pyCO under native conditions; both of these residues are unpaired in the secondary structure model (see Fig. 9). Otherwise, the pattern of strongly reactive sites is dominated

166

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by modification at cytosine residues (e.g. C469, C507 and C518). Unfortunately, a relationship between RNA conformation and reactivity of Et,pyCO with cytosine residues has not been established. We note, however, that the reactive C positions identified in Figure 5 are all unpaired in the secondary st’ructure model. The unreactivity of nearly all adenine residues in this region under native conditions indicates a higher degree of structural organization than is evident, from the secondary structure model. For example, A448, A451, A452. A482 and A487 are all part of an internal loop in the model (see Fig. 9). yet are completely protected under native conditions and only weakly reactive under quasisecondary conditions. The existence of the 455-4621470-477 helix is supportted by our probing data; A456, A459, A460 and A461 are all protected under both native and quasi-secondary conditions. The reactivit’ies of adenine residues in the end loop (positions 463 to 469) of this helix suggests tertiary contacts or a magnesium binding site. Under native conditions, A466 and A468 are protected. while A465 is variably reactive; all of t’hese adenine residues are strongly reactive under quasisecondary conditions. The 437-4401494-497 helix appears to be unstable under our quasi-secondary conditions; A495 and A496. while protected under native conditions, are strongly reactive under quasi-secondary conditions.

(ii) The 1117-1197

region

Only five of the 19 adenine residues in this region show any reactivity toward Et,pyCO under native conditions (Fig. 6); of these. only the weakly reactive A1117 is base-paired in the secondary structure model (see Fig. 9), although in a structure predicted to be only weakly stabilized by stacking interactions (Tinoco et al., 1973). Again, unpaired residues that are unreactive under native conditions but strongly reactive under quasi-secondary conditions are suggestive of tertiary interactions or strong Mg *+ binding sites (A1145, A1157, Al 179 and All&JO). Possible clues to stacking patterns in loops may be suggested also by relative reactivities. For example, All67 was strongly reactive under native conditions in five experiments and weakly reactive in one, while All69 and A1170 were both weakly reactive in five experiments and protected in one. This pattern of reactivity would be compatible with a 3’-stacked configuration for the 1166-l 170 loop, analogous to the configuration of the anticodon loop of tRNA (Kim et al., 1974; Robertus et al., 1974). (iii) The 878-1055

region

In the examples discussed above, and indeed for nearly all of the 309 adenine residues probed (Table 1; Fig. 9), the results of modification under native conditions support the proposed secondary structure model (Woese et al., 1983); i.e. adenine residues that react strongly with Et,pyCO under native conditions are unpaired in the model. Four exceptions are A663, A743, A915 and A918. The reactivity of A91 5 and A91 8 can be seen in Figure 7. These two residues were strongly reactive in all of five experiments. Also noteworthy is the pronounced reactivity of A1042, which appears as a single bulged nucleotide in the secondary structure model (see Fig. 9).

CONFORMATIONAL

PROBING DEN

SEC

OF

167

16 S rRNA

NAT

J

'All88 *llw-

FIG. 6. Autoradiograph of 10qb p 01y acrylamide gel showing Et,pyCO reactivity of nucleotides between positions 1117 and 1191 of 16 S rRNA (cf. Fig. 1, fragment HhaI D). Conditions of modification were as described in Materials and Methods. Positions of specific nucleotides and the xylene cyan01 (xc) marker are indicated. Reactive cytosine residues (not indicated) are found at, positions C1128. Cl147 and C1149. Other designations are as described for Fig. 5.

(iv) The 82-289 drnaturation

region contains

structures

that are unusually

resistant

to thermal

Analysis of adenine reactivities near the 5’ end of 16 S rRNA shows that certain bases are resistant to modification by EtzpyCO even under our denatured conditions (80 mw-potassium cacodylate (pH i’.O), 1 mM-EDTA, 90°C). This can be seen by the prominent gap between Al97 and A238 in the lane representing denatured conditions in Figure 8(a); it is evident that the adenine residues in this region (A199, A205, A223 and A228) are completely unreactive under our denatured conditions. In the region between nucleotides 109 and 279, 13 adenine residues (,4109. A116, A139. A143, A167. A199, A205. A223. A228, A246, A270,

168

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NAT

*1004*1005-

*1042-I

-A1004 %005 *1055-

betw sen I posil ions gel showing Et,pyC’O reactivity FIG. 7. Autoradiograph of a lO”, p 01j wrylamide were as descr ,ibed 860 and 1055 of 16 S rRNA (cf. Fig. 1. fragment AluI E). Conditions of modification in Materials and Methods. Positions of nucleotides and the xylene cyanol (xc) mark, er are in dicatc zd.

(‘ONFORMATIONAL

PROBING

OF

16 S rKSA

I Ii!)

(b) SEC

DEN

A/G

SEC

DEN

I I

A263-

.

1 I I I I I I I I I

A270-

Frc:. 8. Autoradiography between positions 167 and rrspectively). Conditions of under rapid RNA sequencing adrninr residues, described

-I

of a 10 o/b polyacr+nide gel showing E2t,pyC’O reactivit! of nuclrotides 270 of 16 S I-RNA (cf. Fig. 1. fragments $IrtI G and J: Fig. 8(a) and (I)). modification were as described in Materials and Methods. A/U. modified conditions (Peat,tie, 1979). Broken lines bracket a region of unreactive in the text. xc marks the position of the xylene cyanol marker dye.

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A274 and A279) are consistently found to be unreactive and five (A129, A130, A236, A262 and A263) are only weakly reactive under these conditions. The absence of bands in this region cannot be explained by artifacts attributable to the hybridization procedure. For example, under somewhat harsher conditions used for rapid RNA sequencing (50 mM-sodium acetate. 1 mM-EDTA (pH 4*5), 90°C; Peattie, 1979), the missing adenine residues become reactive and produce observable bands (Fig. 8(a), A/G lane). Most striking is A246, which appears strongly reactive in 50 mw-sodium acetate, 1 mM-EDTA, pH 4.5 (A/G lane), but is completely unreactive when modified in 80 mM-potassium cacodylate (pH 7.0), 1 mM-EDTA at 37°C or at 90°C (Fig. 8(a)). The pattern of missing A bands is independent of the length of the isolated fragment. (Compare lanes representing denatured and quasi-secondary conditions in Fig. 8(a) with those similarly marked in Fig. 8(b), which were recovered from hybrids of different lengths, cf. Fig. 1.) Finally, lack of modification is not due to some inherent unreactivity of these bases; when fragments from this region are obtained by hybridization of unmodified RNA and then modified in CEDTA buffer at 9O”C, the previously unreactive adenine residues are found to be reactive (data not shown). We conclude from these results that there is an unusually tight structural organization within the 109-279 region in the intact 16 c”, rRNA. This structure is resistant to disruption. even at 90°C in conditions of low ionic strength and in the absence of divalent cations.

4. Discussion (a) Use of RNA-DNA

hybridization

in chemical probing of RNA

Chemical modification by conformation-specific chemical probes has proven to be a rapid, reliable and specific method for the detailed analysis of higher order structure of RNA (Peattie & Gilbert, 1980; Peattie & Herr, 1981; Douthwaite et al., 1983a,b; de Bruijn & Klug, 1983). A n important advantage of this approach is that the reactivity of each nucleotide, unreactive as well as reactive, is documented. However, a major obstacle to the application of this methodology to the analysis of the structures of large RNA molecules, such as messenger or ribosomal RNA, is the difficulty of clearly resolving nucleotides more than 200 positions from the labeled end. We describe here a general and reliable procedure that extends the method of Peattie & Gilbert (1980) to RNA molecules of any size. The procedure employs the use of RNA-DNA hybridization to generate specific RNA fragments, and is generally applicable to any large RNA for which a cloned DNA coding sequence is available. With this procedure, chemically modified RNA can be cut into specific fragments of predictable size, end-labeled, and the sites of modification identified by gel electrophoresis. Furthermore, we have shown that no significant alteration in the modification pattern is introduced by the hybridization procedure; results are the same if the RNA is modified before or after hybridization. This approach is tailored to the use of chemical probes, such as diethylpyrocarbonate and dimethylsulfoxide, that lead to hydrolysis of the glycosyl bond between the base

CONFORMATIONAL

PROBING

OF

171

16 S rRNA

and ribose. rendering the chain sensitive to aniline-induced strand scission. This permits the use of chemical probes to obtain detailed structural information for large RNA molecules, potentially for every A, G and C residue. An alternative approach, using hybridization with fragments of rRNA genes cloned in the singlestranded phage M13, also permits isolation of specific regions of 16 S rRNA (I). E. I)rapt:r & J. Kean, personal communication). (b) Modi$cation

of cytosine

by diethylpyrocarbonate

During the course of these experiments, we observed aniline-induced chain scissionof Et,pyCO-modified RNA at positions corresponding to cytosine residues (cf. Fig. 5), whereas the chemical specificity of Et,pyCO with RNA has been reported to be limited to adenine and guanine (Peattie & Gilbert, 1980; Dout)hwaite et al., 1983a,b; de Bruijn & Klug, 1983). The probable explanation for t.his apparent anomaly is that reaction at cytosine is greatly potentiated by the relatively high salt concentrations employed for our native reaction condit,ions. High salt concentrations are used to enhance cytosine reactivity with hydrazine in rapid RNA sequencing (Peattie, 1979). Cytidine has been shown to react with Et,pyCO (Vincze et al., 1973), but no such salt dependency has been reported to 0111 knowledge. Although only certain cytosine residues in 16 S rRNA are F:t,pyCO-reactive. it is not clear which structural parameters render them so. They are distributed throughout the rRNA, in both helices and loops of the secondary st#ructure (see Figs 5 and 6), but are found most frequently in singlest,randed regions, at the base of helices, or adjacent to non-Watson-Crick basepairs. Further studies using tRNA. for example, may shed light on the structural requirement for this reaction, which could then be added to the available range of c,onformation-dependent probes. (c) The secondary

structure of naked 16 S rRNA structure in the ribosome

closely resembles

its

The information summarized in Table 1 allows a detailed comparison of the solution conformation of naked 16 S rRNA with a secondary structure model that has been derived mainly from comparative sequenceanalysis (Noller. 1979; Woese Pt (cl.. 1980: Noller & Woese, 1981; Woese et al., 1983). Because of the nature of the comparative approach, and its freedom from complications related to any experimental procedures, we believe the proposed model accurately reflect’s the secondary structure of rRNA in vivo. Thus, while any discrepancy between our c~hemic~al probe findings and the proposed model could be interpreted as evidence against t)he model, they are more likely indicative of differences between the strucsture of naked 16 S rRNA in solution versus its ribosomal conformat’ion. 1nterpret’ation of diethylpyrocarbonate reactivity data rest’s on earlier findings (Peattie & Gilbert, 1980), which show that this reagent reacts only with unpair&l adrninr residues. Thus, any fully reactive sites must be unpaired under our (Jxperimental conditions. Interpretation of the significance of unreactive adenine residues is more complex, however. due to the possibility that tertiary

172

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interactions, stacking, or tight binding of Mg2+ could also shield a base from attack by Et,pyCO. In view of this, the most critical test is that adenine residues shown as base-paired in the model should not be Et,pyCO-reactive. Of the 309 positions probed, in only four cases do we observe reactivity at residues that are involved in pairing (Table 1; Fig. 9). Douthwaite et al. (1983a) have probed the 3’-terminal region of end-labeled 16 S rRNA from three organisms, including E. co&, with Et,pyCO. These authors also noted close accord between their probing results and this part of the secondary structure model. Our findings, insofar as we have probed this region of the molecule (Table l), are in generally good agreement with theirs, with the exception of positions 1363, 1394 and 1447. Residues 1363 and 1447 are more reactive in the studies reported by Douthwaite et al. (1983a) than in those reported here; residue 1394 is more reactive in our hands than in theirs. Unreactivity at position 1363 may be rationalized by pairing of A.U,,,s with A*U1364 in our RNA preparation (a pairing that is not supported by comparative analysis and so is unlikely to exist in the ribosome). Agreement with the model is therefore extremely good, supporting its validity on the one hand, as well as pointing out the close similarity of the secondary structures of 16 S rRNA in solution and in the ribosomes, on the other. The four discrepant positions, A663, A743, A915 and A918, are of interest. A663 is involved in a suggested, but phylogenetically unproven, A *G “pair” in the model (Fig. 9); possibly, this is part of the structural feature that requires interaction with ribosomal proteins to attain its functional conformation. In this connection, it has been shown that the adjacent nucleotide, G664, is reactive toward kethoxal in the isolated RNA containing the binding sites for proteins 58 and S15 (Miiller et al., 1979). When 515 is bound to the RNA, G664 becomes shielded from kethoxal, suggesting either that the protein binds directly to this region of the RNA, or that it induces a conformational change involving G664. Because of its proximity, A663 could also be part of either process. Reactivity of A743, opposite A663 in the same helix, similarly may be related to binding of S15: the neighboring nucleotide G742 also losesits kethoxal reactivity when S15 binds (Miiller et al., 1979). The remaining two anomalously reactive adenine residues (A915 and A918) are found in a very interesting structural feature, the 1720/915-918 helix. This helix is the sole example documented thus far in the rRNAs where nucleotides in a loop are found to pair with nucleotides outside of the helix that defines the loop (in this case, the g-13121-25 helix). We previously noted disruption of this helix in naked 16 S rRNA (Woese et aZ., 1980), iondepleted E. coli 30 S subunits (Hogan & Noller, 1978), and yeast 40 S subunits (Hogan et al., 1984), suggesting that this tertiary-like feature of the structure may be particularly fragile. (d) Evidence

for speci$c

tertiary

interactions

Adenine residues that are not base-paired in our secondary structure model but are nevertheless resistant to modification by Et,pyCO under native conditions must either be part of some, as yet unidentified, helix, or involved in tertiary structure or a strong magnesium ion binding site. So little opportunity for

(‘ONFORMATIONAL

PROBING

OF

16 S rRNA

FIG. 9. Secondary structure model of E. coli 16 S rRNA (Woese et al., 1983) showing Et,pyCO reactivity of specific adenine residues under native and quasi-secondary conditions. Reactivity under native conditions is denoted by circles: 0, unreactive; 0, weakly reactive; and 0, strongly reactive. Reactivity under quasi-secondary conditions is denoted by triangles: A, unreactive; A, weakly reactive; and A, strongly reactive. Conventional sequence hyphens have been omitted. Centre dots indicat,e G U or A. G base-pairs, while hyphens indicate G. C or A. U base-pairs.

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additional pairing exists in the model (Fig. 9) that it is unlikely that many helices remain undiscovered; thus, most of the adenine residues belonging to the aforementioned class are more likely t,o be protected as a result of tertiary or magnesium ion contacts. Further insight is provided by comparison of relative reactivities under different ionic conditions. Studies on tRNA have shown that tertiary structural features tend to be less stable, and more strongly dependent on ionic conditions, particularly Mg2 + , than is secondary structure (for a review, see Crothers, 1979). Peattie & Gilbert (1980) exploited this relationship, and were able to distinguish most of the tert,iary structural features of yeast tRNAph’ from secondary structure by chemical probing under controlled ionic conditions. It is reasonable to expect that much of the tertiary structure of ribosomal RNA will behave in a similar fashion. Tndeed, studies on ion-depleted 30 R ribosomal subunits showed that they possess many more kethoxal-reactive guanine residues than do normal 30 S subunits, and t’hat virtually all of the newly reactive sites are in unpaired regions of the 16 S rRNA secondary structure model (Hogan & Noller, 1978). Here, we have followed the approach used by Peattie & Gilbert (1980), monitoring the reactivity of adenine residues in 16 S rRNA in the presence of Mg2 + and high salt (native conditions) or in EDTA/low salt (quasi-secondary conditions) at 37°C. Our results clearly show that 16 S rRNA has a more open conformation under quasi-secondary conditions than it does under native structure conditions. In the native structure, only 14% of the adenine residues are strongly reactive as compared to 52% under quasi-secondary conditions (see Table 2). Partial reactivity is also increased from So/b for native to 18% for quasisecondary, which we take as evidence of a greater degree of structural heterogeneity in the quasi-secondary case. Likely candidates for tertiary interaction would include adenine residues that are (1) unreactive under native conditions, (2) reactive under quasi-secondary conditions, and (3) unpaired in the secondary structure model. By analogy with the findings of Peattie & Gilbert (1980), nucleotides involved in tight binding of Mg2 + will also fit these criteria. Examination of the results summarized in Table 1 and Figure 9 shows that there are 57 adenine residues of this class. or about 20% of the total number of nucleotides examined. This compares reasonably well with TABLE

Summary

2

of modi$cation

results

Modification conditions

Strongly reactive

Weakly reactive

Unreactive

CMK (37°C) CEDTA (37°C)

44 (14%)

25 (So/b)

240 (78%)

309

126 (52%)

44 (18%)

72 (30%)

242

Conditions of modification were as described in Materials from the total number of adenine residues examined under number of adenine residues examined under each condition.

and Methods. the condition

Total

Percentages stated. Total

are calculated represents the

CONFORMATIONAL

PROBING

OF

16 S rRNA

Ii5

the corresponding number of nucleotides in tRNAphe that are unpaired in the cloverleaf scheme and are involved in tertiary interaction (16 out of 76, or also about 20%: Rich k RajBhandary, 1976; Kim, 1976; Jack et aE., 1976). This catalogue of tertiary structural candidates may prove useful in future attempts to discover specific features of the three-dimensional structure of 16 S rRNA. Adenine residues in base-paired regions of the secondary structure model that are reactive under quasi-secondary conditions could be interpreted either as evidence against the model, or as weak secondary structure. We find 29 such reactive adenine residues (Table l), 12 of which are at the ends of helices and 17 of which occur at internal positions. In each case, where we find strongly reactive adenine residues in the middle of a helix, the data are consistent with t)he interpretation that the entire helix is unravelled; i.e. in no case do we observe a residue in the middle of a helix to be fully reactive while another residue in t’he same helix is protected. Some of these unstable helices contain an abundance of A .I: and/or G ’ U base-pairs, which may account for their instability under these conditions. Consider, for example, the 1118-1124/1149-1155 helix; out of seven base-pairs in this helix, four are A. U. In the 1006-1012/1017-1023 helix, four base-pairs out of a total of seven are A. U, and two are G * U. Both of these helices appear unstable under our quasi-secondary conditions. In conclusion, our findings suggest that, while most of the helices remain intact under quasi-secondary condit#ions, some tend to fray at the ends, and a few become completely disrupted. I$Te identify 70 adenine residues that are protected from modification under bot,h native and quasi-secondary conditions; 39 of these are found in helices in t’he secondary structure model and 31 are found in single-stranded regions (Fig. 9). Adenine residues in the latter group may be involved in helical structure not yet verified by phylogenetic evidence, or in unusually stable tertiary structure. Finally, we have discovered an intriguing class of adenine residues between positions 109 and 279 of 16 S rRNA that are unreactive to Et,pyCO even under our denaturing conditions (80 mM-potassium cacodylate (pH 7*0), 1 mM-EDTA. 90°C’: Fig. 8). Examination of the secondary structure model in t,his region fails to suggest any obvious structural basis for this unusual thermal stability; although extensive base-pairing is found here, it does not appear to differ qualitatively in its predicted stability (Tinoco et al., 1973) from more labile structure found elsewhere in the molecule. One possible contribution to stability here is a potential coaxial stacking arrangement (Woese et al., 1983) that can he envisaged for this region; this is generated by stacking the 122-128/233-239 helix on the 240-2451281-286 helix, analogous to the stacking of the aminoacyl and TW’ st,ems of t,RNA (Kim et al., 1974; Robertus et aE., 1974). By invoking some irregular pairing and/or looping, this results in a more or less continuous series of stacked helices stretching from position 210 to 260, which would comprise the aforrment’ioned thermostable residues. It is notewort,hy that this region is the heart of a proposed RNA “core”, a nuclease-resistant struct’ure that includes much of the 5’ domain of 16 S rRNA (Garrett et al., 1977). Perhaps its st’ability is related to formation of a nucleation site for ribosome assembly. Tn this study, we have demonstrated the utility of our approach in probing the higher-order structure of ribosomal RNA. An obvious extension of this approach

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is to monitor structure-function relationships in the ribosome, as has been done earlier using kethoxal, for example (Herr et aZ., 1979). Furthermore, this method should be readily applicable to the study of other classes of large RNAs, such as messengers and their precursors. We thank R. Garrett, R. Ogden and D. Peattie for advice on chemical modification, 8. Douthwaite. R. Gutell, J. Prince, S. Stern, K. Triman, V. Wheaton and L. Zagorska for helpful comments on the manuscript, and J. Normanly for construction of pJN53. This work was supported by grant no. GM-17129 from the I1.S. National Institutes of Healt,h. REFERENCES Brimacombe, R., Maly, P. & Zwieb, C. (1983). Prog. Nucl. Acid Res. Mol. Biol. 28, l-48. Brosius, J., Palmer, M. I,., Kennedy. P. J. & Noller. H. F. (1978). Proc. ~Vat. Acad. Sri., C’.S.A. 75, 4801-4805. Brosius, J., Dull, T. J., Sleeter, D. D. & Noller, H. F. (1981a). J. Mol. Biol. 148, 107-127. Brosius. J.. Ullrich. A.. Raker, M. A.. Gray. A.. Dull. T. J.. Gutell. R. R. & Noller. H. F. (19816). PZasmid, 6, 112-l 18. Rruce. A. G. & Uhlenbeck, 0. C. (1978). Nucl. Acids Res. 5, 3665-3677. Casey, J. & Davidson, N. (1977). Nucl. Acids Res. 4. 1539-1552. Crothers, D. M. (1979). In Transfer RNA: Structure, Properties, and Recognition (Schimmel, P. R., Soll. 1). 8r Abelson.
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