,I. Mol. Bio2. (1990) 211, 699-712

Structural Analysis of the Internal Transcribed Spacer 2 of the Precursor Ribosomal RNA from Saccharomyces cerevisiae Lee-Chuan C. Yeh and John C. Lee The University

Department of Biochemistry of Texas Health Science Centre at San Antonio TX 782847760, U.S.A.

(Received 8 August

1989; accepted 3 October 1989)

Full-length precursor ribosomal RNA molecules (6440 bases) were produced in vitro using a plasmid containing the yeast 35 S pre-rRNA operon under the control of phage T7 promoter. The higher-order structure of the internal transcribed spacer 2 (ITS-2) region (between the 5.8 S and 25 S rRNA sequence) in the pre-rRNA molecule was investigated using a combination of enzymatic and chemical structural probes. The data were used to evaluate several structural models predicted by a minimum free-energy calculation. The results supported a model in which the 3’ end of the 5.8 S rRNA and the 5’ end of the 25 S rRNA are hydrogen-bonded better than the one in which the ends are not. The model contains a high degree of secondary structure with several stable hairpins. Similar structural models for the ITS-2 regions of Schizosaccharomyces pombe, Saccharomyces carlsbergensis, mung bean and Xenopus laevis were derived. Certain common folding features appear to be conserved, in spite of extensive sequence divergence. The yeast model should be useful as a prototype in future investigations of the structure, function and processing of pre-rRNA.

1. Introduction In Saccharornyces cerevisiae, the primary 35 S precursor rRNA has the following sequence organization: 5’ LETS?/18 S rRNA/ITS-l/58 S rRNA/ ITS-2/25 S rRNA/3’TETS (Skryabin et aZ., 1979a,b; Bayev et al., 1981). The leader external transcribed spacer (LETS) is about 650 nucleotides. The internal transcribed spacer 1 (ITS-l) is 360 nucleotides; the internal transcribed spacer 2 (ITS-2) is 235 nucleotides. The terminal experimental transcribed spacer (TETS) varies between 7 and 200 nucleotides (Skryabin et al., 1979a,b; Planta & Raue, 1988; Warner, 1989). The 3’ end of the 35 S pre-rRNA is generated by endonucleolytic cleavage followed by exonucleolytic cleavage of about seven nucleotides (Veldman et al., 198Ob). The 35 S prerRNA is processed to two RNA intermediates, the 27 S and 20 S species (Warner, 1982). The 27 S RNA intermediate is further processed to give rise to the mature 25 S and the 5.8 S rRNA species. The

t Abbreviations used: LETS, leader external transcribed spacer; ITS, internal transcribed spacer; TETS, terminal external transcribed spacer; DMS, dimethylsulfate; CMCT, 1-cyclohexyl-3-(2morpholinoethyl)-carbodiimide metho-p-toluenesulfonate. 0022-2836/90/04069%14

$03.00/O

20 S RNA intermediate is processed to the 18 S rRNA. All four RNA spacers are removed during processing. Whereas the structures of the mature rRNAs are rather well conserved, the structures of the transcribed spacers appear to be divergent (Verbeet et al., 1984; Gerbi, 1985). Although the primary structure of the pre-rRNA for several organisms has been determined for some time, very little is known about their secondary and tertiary structure. Nonetheless, several secondary structural models have been predicted on a theoretical basis (Hall & Maden, 1980; Jordan et aZ., 1980; Veldman et aZ., 1980a, 1981; Bayev et al., 1981; Schaak et al., 1982; Subrahmanyam et al., 1982; Furlong & Maden, 1983; Michot et al., 1983; Hindenach & Stafford, 1984; Nazar et al., 1987). Based solely on terminal nucleotide sequence information of the ribosomal processing intermediates, Veldman et al. (1981) postulated that small hairpin structures around the processing sites may be involved in the maturation of the yeast precursor RNA. Analysis of the higher-order structures of these pre-rRNA molecules may shed light on the processing mechanism and ribosome biogenesis. Recently, we successfully cloned the yeast rRNA gene in an expression vector and produced the yeast 35 S pre-rRNA in vitro. Initial studies on the structure of the RNA species in solution provided data

699

0 1990 Academic Press Limited

L.-C. C. Yeh and J. C. Lee

700

for a secondary structure model for the ITS-l region (Thweatt & Lee, 1989). Studies from other laboratories on the in vitro Escherichia coli pre-rRNA molecule lend credence to the idea that the synthetic RNA species are similar to the native molecules. The synthetic RNA molecule can bind ribosomal proteins (Mougel et al., 1987; El-Baradi et al., 1987), form part of a functional ribosome (Krzyzosiak et al., 1987; Melancon et al., 1987), undergo correct processing (Stern et al., 1988), and can be methylated correctly in vitro (Negre et al., 1989). Here we report studies on the higher-order structure of the ITS-2 sequence within the 35 S pre-rRNA using a combination of enzymatic and chemical structural probes. These results provided a detailed experimental evaluation of a model, which was derived from minimum free-energy calculations for the ITS-2 region. Data obtained agreed well with the proposed model provided that the 3’ end of the 5.8 S RNA sequence base-pairs with the 5’ end of the 25 S RNA sequence bringing the ends of the ITS-2 sequence together. Similar structural models for the ITS-2 regions of Schizosaccharomyces pombe, Saccharomyces carlsbergensis, mung bean and Xenopus laevis have been derived. Certain common folding motifs are observed, though the primary structures are divergent. The yeast model should be useful in further investigations of the structure, function and processing of pre-rRNA and ribosome biogenesis. 2. Materials

and Methods

(a) Buffers

Transcription buffer (5 x ): 100 mm-sodium phosphate (pH 7.9), 250 IUM-&Cl, 40 mM-Mgcl,, 50 mM- dithiothreitol, 4 mnn-spermidine. DNase digestion buffer (5 x ): 200 mM-Tris*HCl (pH 7.5), 50 mM-&Cl, 30 mM-Mg&, 50 mM-dithiothreitol. Gradient buffer: 31 y0 to 34% (w/v) sucrose, 40 mM-Tris (pH 12), 20 mm-acetic acid, 2 mM-EDTA, @5% (w/v) sodium dodecyl sulfate. CMK: 80 mM-potassium cacodylate (pH 7.2), 20 mM-magnesium acetate, 300 mM-KCl. CE: 80 mM-potassium cacodylate (pH 7.2), 1 mM-EDTA. BMK: 50 mM-potassium borate (pH %O), 20 mm-magnesium acetate, 300 mM-Kcl. BE: 50 mna-potassium borate (pH 8.0), 1 mM-EDTA. DMSstop buffer: 1 M-Tris-acetate (pH 7.5), 1.5 M-SOdiUUI acetate 0.1 mM-EDTA, 1 M-p-inercaptoethanol. Polynucleotide kinase buffer: 250 miw-Tris. HCl (pH 7.5), 25 mM-Mgcl,, 50 mM-fi-mercaptoethanol. Oligonucleotide elution buffer: 45 mM-Nacl, 2.5 m&sodium phosphate Hybridization buffer (5 x ): (PH 7)> 1 mM-EDTA. 250 mM-potassium-Hepes 500 mM-KCl, (PH 7)> 25 mlvr-potassium borate. Primer extension buffer (5 x ): 250 mM-TriseHCl (pH &5), 250 mi%-KCl, 50 mm-M&l,, 50 mM-dithiothreitol, @5 mM each of dCTP, dGTP and

dTTP. Chase buffer: 1 mM each of dATP, dCTP, dGTP and dTTP. Reverse transcriptase buffer: 50 mM-Tris . HCl (pH %5), 50% (w/v) glycerol, 2 mM-dithiothreitol. Sample loading buffer: @03% (w/v) each of xylene cyanol and bromophenol blue in deionized formamide. Gel electrolyte buffers: upper chamber = @5 x TBE (1 x TBE: 89 mM-Tris, 89 mnr-boric acid, 2 mM-EDTA, pH &3), lower chamber = @67x TBE, 1 M-sodium acetate.

(b) In vitro transcription The plasmid (pD) containing

the rDNA

coding for the

35 S precursor rRNA in pGEM-2 (Thweatt & Lee, 1989) was linearized with PstI. About 2 pg of the DNA template was treated with Klenow DNA polymerase (Promega, 60 units) at 22°C for 30 min to remove the 3’ overhang structure. The repaired template was transcribed in vitro in a 100 pl reaction mixture containing 1 x transcription buffer, 1 mM each of ATP, CTP, GTP and TTP, 160 units RNasin (Promega) and 400 units phage T7 RNA polymerase (New England Biolabs) at 37°C for 60 min. The reaction was stopped by phenol/chloroform extraction and precipitation with ethanol. The template was removed by digestion with 25 units of DNase I (Promega) in 1 x DNase digestion buffer at 37°C for 30 min. After phenol/chloroform extraction, the pre-rRNA was recovered by precipitation with ethanol and purification through a 31 y. to 34% sucrose gradient at 18”C, 24,000 revs/min for 16 h in a Beckman SW 50.1 rotor. A portion (10 ~1) of each fraction was analyzed on a 1 y. (w/v) agarose gel in 10 m&sodium phosphate (pH 7.9). The reservoir buffer was circulated during electrophoresis. Fractions containing the pure pre-rRNA were pooled and precipitated with ethanol. (c) Pretreatment of pre-rRNA For “native” conditions, the pellet containing 1 pmol pre-rRNA was dissolved in 100 ~1 of CMK buffer (for enzymatic digestions or modification with dimethylsulfate) or BMK buffer (for modification with carbodiimide). The sample was incubated at 42°C for 20 min, cooled slowly to room temperature for renaturation, and placed on ice for 5 min. For denaturing conditions, the pellet was dissolved in CE buffer (for DMS modification) or BE buffer (for CMCT modification). For each type of modification, a control RNA sample was run simultaneously under identical conditions except omission of the enzyme or the modifying chemical agent. (d) Chemical modijcations Conditions for chemical modification of yeast pre-rRNA were basically similar to those described previously with minor changes (Moazed et al., 1986; Thweatt & Lee, 1989). (i) Dimethylsulfate (Aldrich Chem. Co.) modijication For “native” conditions,,!pre-rRNA was incubated with DMS (0.35 ~1 DMS/pmol RNA) at 25°C for 15 min. For denaturing conditions, the modification reaction was performed at 90°C for 1 min with 2 to 4-fold less DMS. The modification reaction was terminated by the addition of 025 vol. DMS-stop buffer and 2.5 vol. ethanol. (ii) Carbodiimide (Sigma) modijcation For “native” conditions, pre-rRNA was incubated with CMCT (84 pg CMCT/pmol RNA) at 25°C for 15 min. For denaturing conditions, the reaction was carried out at 90°C for 1 min with 2 to 4-fold less CMCT. The modification was stopped by adding @l vol. 3 M-sodium acetate and 2.5 vol. ethanol. (e) Enzymatic modi$cations Conditions for enzymatic modifications were similar to those described by Ehresmann et al. (1987). Pre-rRNA was digested with RNase T, (Calbiochem, 0002 unit/pm01 RNA) or RNase V, (Pharmacia, 001 unit/pmol RNA) at

Yeast Precursor Ribosomal RNA 25°C for 10 min. Both reactions were stopped by extraction with phenol/chloroform and precipitation with sodium acetate and ethanol. (f)

Synthesis

and

labeling

of oligonucleotide primers

Three 17-mers were synthesized using an Applied Biosystem model 3808 DNA synthesizer. Primers A, B and C are complementary to residues 53 to 69, 128 to 144 in the lTS-2, and 7 to 23 in the 25 S RNA sequence, res ectively. Primers were labeled at the 5’ end with [y- P, P]ATP and phage T4 polynucleotide kinase. Labeled primers were purified on 20% (w/v) polyacrylamide gels in 1 x TBE buffer. Following autoradiography, the gel piece containing the labeled primer was excised and incubated in the oligonucleotide elution buffer at 37°C overnight. The radioactive elute was recovered and a portion (about 50,000 cts/min) was used for each annealing reaction. (g) Primer annealing

and extension

Modified and unmodified pre-rRNA samples were annealed in 1 x hybridization buffer (3 ~1) with a primer at 90°C for 2 min and cooled slowly to 45°C. Extension reaction (Moazed et al., 1986) was carried out at 28°C for 30 min in 1 x extension buffer (5 ~1) containing 200 PM-dATP and AMV reverse transcriptase (2 units, Promega). For the modified samples, chase buffer (2 ~1) was included in the reaction mixture. For the unmodified dideoxy sample, the corresponding dideoxy nucleotide was added instead. After 30 min, the chase buffer was added and the reaction was incubated for an additional 15 min. The extension reaction was stopped by the addition of 3 M-sodium acetate and ethanol. Pellets were dissolved in 3 ~1 of sample loading buffer, incubated at 65 ‘C for 3 min, and applied to an 8% (w/v) thin polyacrylamide gel. Electrophoresis was carried out in gel electrolyt,e buffers at’ constant power (20 W) for 3 h. The gel was tixed in methanol/acetic acid/water (10 : 10 : 80, by vol.) for 15 min, dried under vacuum, and subjected to autoradiography. For each primer used, experiments were performed at least 3 to 4 times with different preparations of pre-rRlu’A. (h)

(‘ompu,ter

analysis

of RNA

structure

The Sequence Analysis Software Package (version 6) for the VAXVMS computer produced by the Genetics

Computer Group (GCG) of the University of Wisconsin was used to analyze the secondary structure of the ITS-2 region. The program FOLD, which finds a best fit secondary structure with minimum free-energy (Freier et al., 1986) for a RNA molecule by the method of Zuker & Stiegler (19X1), was used.

3. Results (a) Reactivity of bases with chemical reagents Reactivity of bases in the ITS-2 sequence within the 35 S pre-rRNA was probed with chemical reagents. Two different chemical probes (DMS and

CMCT) that under

“native”

react with conditions

unpaired

bases at 25°C

were used. Modifications

were also carried out at 30°C under denaturing conditions. Dimethylsulfate reacts with adenine at N-l and more slowly cytosine at N-3; CMCT reacts

701

with uracil at N-3 and more slowly guanine at N-l (Ehresmann et al., 1987 and references cited therein). The reactive bases were detected by primer extension with reverse transcriptase (Stern et al., 1988). Three

DNA

primers,

spaced along the RNA

molecule, were used in order to provide overlapping information on the reactivity of bases within the entire ITS-2 sequence. The base sequence of each primer is complementary to a specific sequence in the pre-rRNA.

The identity

of the modified

bases

was facilitated by running the appropriate dideoxy sequencing lane on the same gel. Artifact bands were distinguished from modified sites by their presence in DNA molecules using unmodified control pre-rRNA. Representative autoradiograms from several experiments in which the DMS and CMCT modifications were carried

out under

native

(Figs

1 and 2)

and denaturing (Figs 3 and 4) conditions are shown. Results are summarized in Tables 1 and 2. A comparison of the reactivity of adenine residues towards DMS under the native and denaturing conditions revealed that most of the unreactive bases became reactive upon denaturing of the RNA molecule, e.g. adenine residues 8 to 10, 24, 28 and 32 (Table 1). However, adenine residues 127 and 171 were not modified even when the RNA was denatured. This apparent lack of reactivity was more pronounced for cytosine residues (Table 1). This was expected because it has been shown that DMS reacts with cytosine many-fold slower than with adenine. The pattern of reactivity of CMCT with uracil residues in the native and denatured form of the RNA is shown in Table 2. Except for uracil residues 65 to 68 and 71 to 72, all unreactive uracil residues in the native RNA molecule became reactive upon denaturation of the RNA.

(b) Reactivity of bases as probed by enzymes Enzymatic probing was achieved with R’Nase T, and V, on the ITS-2 region of the pre-rRNA in the native state. The former enzyme will cleave unpaired guanine residues and the latter paired or stacked nucleotides. Cleavage sites were detected by primer extension with reverse transcriptase. Representative autoradiograms are shown in Figures 1 and 2 and the results are summarized in Tables 3 and 4. When the pre-rRNA was in the native condition, very few guanine residues in the ITS-2 sequence were cleaved by RNase T, (Table 3). Table 4 shows the data on RNase V, digestion of native pre-rRNA. The results are in reasonable agreement with the data on the DMS and CMCT reactivity of the corresponding residues in the denatured pre-rRNA, i.e. residues that are cleaved by V, in the native RNA molecule are not reactive with DMS or CMCT unless the RNA is denatured. Results of the chemical and enzymatic probing of the ITS-2 sequence within the yeast pre-rRNA molecule are superimposed on a structural model for the ITS-2 region (Fig. 5). The model was generated

L.-C. C. Yeh and J. C. Lee

702

v, 012

Tl

Gi-5

DMS A12

CMCT lJ12

-Cl30

-G'150

-Cl

-u20

-u40

Fig. 1.

703

Yeast Precursor Ribosomal RNA

Table 1 (continued)

Table 1 Modification of the ITS-2 region under native and denatured conditions with dimethyl sulfate

Adenine

8-16

Native

Denatured

1 2 5 I

++ -

++

11

++ ++ -

Cytosine

12 15 24 2x 32 34 43 47-48 49 53-55 59 63-64 69 70 75 88-89

115 119 121-122

102 104 107 111

125

126 127 I30 131 135 141 148

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

149-150 151-152

+ 153 156 159

161-162 164 171 173 174 177 182 187-188

179

++ -

Cytosine 189

++ 4-f f + + ++ + ++ + ++ + ++ + + + + ++ ++ ++ + + ++ + ++ ++ ++ + + + + + + + + + + + + ++

Native

-

193

-

++ 35 37

W-92 94 96

Adenine

Condition

Residue

Condition

Residue

195 197 199 200 202-203 205 207 209 212 214 217 219-220 221 222-223 228 231 232 233

++ ++ + + + ++ + -

Denatured + + + ++ ++ ++ ++ ++ + ++ +-I++ ++ ++ + ++ ++ ++

Pre-rRNA was modified under native or denatured conditions with DMS. Modified residues were detected by primer extension. DMS reacts with unpaired adenine primarily but also with cytosine slowly; + + , residue reacted with DMS strongly; + , reacted weakly; -, not reactive. The number refers to the residue position in the ITS-2 sequence.

by theoretical minimum free energy calculation (AG = - 1085 cal/mol; 1 cal = 4.184 J) and by consideration of potential contributions from the 50 bases from the flanking 25 S rRNA sequence and the entire 158 bases from the 58 S rRNA sequence. A different model was generated when only the ITS-2 sequence was considered. The experimental data fit the former model much better than the latter (78% versus 49 oh of the residues experimentally shown to be in either base-paired or singlestranded configuration were situated in the correct configuration in the model).

4. Discussion Experimental results were obtained from chemical and enzymatic probing of the ITS-2 sequence within the yeast pre-rRNA molecule. These data agreed better with the theoretical structural model for the ITS-2 region in which the 3’ end of the 5.8 S rRNA and the 5’ end of the 25 S rRNA are hydrogen-bonded than one that are not. Our results do not support the computer-generated model for S. cartsbergensis (Nazar et al., 1987), even though

Figure 1. Autoradiograms showing sites within the ITS-2 sequence that were chemically or enzymatically modified under native conditions using primer A. Four modifiers were used: V,, T,, DMS and CMCT. Lane 0 is the unmodified control that was treated as the modified samples except for omission of reagent. For each modifier, a control sample with unmodified pre-rRNA was run by dideoxy sequencing, generated by reverse transcription in the presence of ddCTP, ddTTP and ddATP (lanes G, A and U for T,, DMS and CMCT, respectively). Two concentrations of each modifier were used (lanes 1 and 2). For V,, the concentrations were @Ol and O-02 unit/pm01 pre-rRNA. For T,, @002 and O+lO4 unit/pmol. For DMS, 035 and 0’70 pl/pmol. For CMCT, 84 and 168 pg/p mol. Positions of the corresponding nucleotides are denoted on the right. Cl is the first residue of ITS-2 and residues with a prime are from the 58 S rRNA sequence.

L.-C. C. Yeh and J. C. Lee

704

CMCT U12

-G190

-G210

-U230

Figure 2. Autoradiograms under

native

conditions

showing sites within using primer C. Conditions

the ITS-2 sequence that and symbols are similar

were chemically or enzymatically to those used for Fig. 1.

modified

705

Yeast Precursor Ribosomal RNA

Table 3 Modi$cation

Table 2 of the ITS-Z region under native and denatured conditions with curbodiimide

Modi$cation

Condition ITridine

Native

3-4

++ ++ ++ + ++ + ++ + + + + + ++ + ++ -

6 13-14 16

1U-20 23

26 30 33 36 38

39-40 45 46

50-51 56-57 60 65-68 71-72 76 78-83 84-85 86-87

99.-101 103 105 109 112 113 118 120 123 129 134 137-139 140 144 145 146 147 154 160 163 165-170

Ii2 17.5 181 183-184 191-192 194 198 211 213 224 226-227

229-230 235

++ ++ + -

Denatured + + + + + + + + + + + + + + + + + + + ++ + ++ ++ ++ ++ ++ ++ ++ ++ ++ + + + + + + + + + + + + + + + + + + + + + + + + +

Pre-rRNA was modified under native or denatured conditions with CMCT. Modified residues were detected by primer extension. CMCT reacts with unpaired uridine residues; + +, residue reacted with CMCT strongly; + , reacted weakly; - , not reactive. The number refers to the residue position in the ITS-2 sequence.

Guanine 17 21-22 25 27

29 31 41-42 44 52 58 61 62 73-74 77

93 95 97 98 106

108 110 114 116-117 124 128 132-133 136 142-143 155 157-158 176 178

180 185-186 190 196 201 204 206 208 210 215-216 218 225 234

the ITS-2 region with RNase T, under native conditions of

Reactivity + + + + f-t +

Pre-rRNA was digested under native conditions with RNase T, Modified residues were detected by primer extension. RNaae T, cleaves unpaired guanine residues; + + , residue reacted with T, readily; +, reacted weakly; -, not reactive. The number refers to the residue position in the ITS-2 sequence.

the primary sequences of the ITS-2 regions of the two yeasts differ only by one residue. A large number of residues experimentally determined to be reactive with single-strand-specific probes is located in double-stranded regions of their proposed model. For discussion, our cross-like model involved basically a long stem with three small hairpin loops, divided into six regions (I to VI). Each substructure will be discussed in detail.

(a) Substructure I This substructure contains a helical encompassing the 3’ end of the 5.8 S rRNA

structure sequence

L.-C. C. Yeh and J. C. Lee

706

DMS OA123

CMCT Ul 23

-U60

-US0

-UlOO

Fig. 3.

Yeast Precursor

Table 4 Mod$cation

of the ITS-2

region with RNase under native conditions

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

(’ (’ 1’ (: C IT (: (: 1’ t: (’ I’ I (: 1 (’ IT 1’ (’ IT (’ (: I(: (’ t: (’ (: (’ IT

(c) Substructure

I’re-rRNA was digested under native conditions with Y, Modified residues were detected by primer extension. V, cleaves base-paired or stacked nucleotides; + +, reacted st,rongly; +, reacted weakly. The number refers residue position in the ITS-2 sequence.

RNase RNase residue to the

(residues 139 to 156) and the 5’ end of the 25 S rRh’A sequence (residues 1 to 18). The majority of residues were susceptible to RNase V1 under the native conditions and not reactive towards singlestrand-specific chemical reagents. These residues became reactive following denaturation of the RNA molecule. The reactivity of Ul was unexpected; it did not react with single-strand-specific probes but became reactive upon denaturation of the RNA. (b) Substructure This substructure 3’ end of the 5.8 S ITS-2 sequence) with end of the stem. The

Figure

1I

consists of residues RNA sequence) to a stem and a small majority of residues

3. Autoradiograms

comparing

107

RNA

stem structure were not reactive with either chemical and enzymatic single-strand-specific probes and became reactive upon denaturation of the RNA molecule. This observation would support the conclusion that these residues are involved in basepairing in the non-denatured RNA molecule. This is further confirmed by the observation that these residues were cleaved by the double-strand-specific RNase V,. Although residues U157, Cl and U6 were predicted to be involved in base-pairing according to the model, they were accessible to DMS and CMCT. The reactivity of U157 and 176 might be explained by the fact that U157 * G29 and lJ6. G22 are non-canonical base-pairs and located at the end of a, stem. Cl is located adjacent to a bulge. Previous data indicate that such interactions are likely weak (Tinoco et al., 1973). Every residue (Al2 to U16) located in the loop region was reactive with single-strand-specific probes.

VI

Reactivity

Residue

Ribosomal

157 (of 29 (of loop at within

sites within

the bhe the the

III

Residues in the single-stranded region were reactive to varying degrees, e.g. U30 was weakly reactive and U33 and A34 reacted strongly. A helical structure was formed between residues 34 to 60 and 215 to 235. The sequence comprising residues 227 to 235 is the 5’ processing site in producing the 5’ end of the 25 S rRNA molecule (Veldman et al., 1981). The cleavage site (C,) suggested by Veldman et aE. (1981) is located at the foot of a relativelv unstable stem with an estimated AC value oi +2 kcal (Tinoco et al., 1973). The two uracil residues, U235 and Ul (of the 25 S rRNA sequence) became reactive only after denaturation. The apparent anomalous unreactivities are not easily explained. Perhaps these residues are involved in tertiary interactions or in an alternative conformation, since they are located at the processing site. Two major bulges (U46-C49 and A53-A55) were located in the middle of the helix. Residues within these bulges were highly reactive with single-strandspecific probes. Roth bulges contained a number of adenine residues; the significance of this is not clear at the present. Residues within the remainder of the helix were either accessible to RNase V, (e.g. C217 and G218) or became accessible t,o single-strandspecific probes upon denaturation of t,he RNA molecule (e.g. U50 to 51 and C221 to lJ224). (d) Substructure

I J’

This stem-loop region is composed of residues 61 to 100. The stem region, consisting of residues 61 to

the ITS-2 sequence of pre-rRKA

that’ were chemically

modified

under nativ? and denatured conditions using primer B. Lane 0 is the unmodified RNA control under native conditions. Lanes A and U are dideoxy sequencing lanes, generated by reverse transcription in the presence of ddTTP and ddATP, respectively. Lanes 1 are modified RNA under native conditions. Two concentrations of each modifying reagent were used under denatured conditions (lanes 2 and 3). For DMS, @09 and 018 ,ul/pmol. For CMCT, 21 and 42 pg/pmol. Nucleotide positions are indicated on the right. The number of bands in lane 3 (DMS) is small compared to that in the other lanes. presumably because at the higher DMS concentration, a larger number of residues were modified so the cDP;A products were shorter.

L.-C. C. Yeh and J. C. Lee

708

MS OA123

CMCT Ul 23

.G190

-G21O

-0230

Figure under

;rams comparing sites within using primer and danatl xed conditions

4. Autoradiog

native

mndified the ITS-2 sequence of pre-rRKA that were chemically > those used for Fig. 3. cf. Conditions and symbols are similar tc

UA A:

ue40

23WU

D 0

AC A0

220eA

70 .a CA uuc Mk @@O c90

80

IV

UO GO AO

OG

P.G FE?-G A&-120 170

C-G .U @A *A

u Acjl30 Co

160W4Q.G K-G G.U G-C C-G G.W

OA+g

CA-U0140 A0 150-+G G OA @ ." U. U

Figure 5. A proposed secondary structural model for the ITS-2 region of the pre-rRNA from S. cerewisiae. The reactivities of individual nucleotides to chemical and enzymatic probes under native and denatured conditions are indicated. **, The beginning of the ITS-2 sequence; *, the beginning of the 25 S rRNA sequence; -, Watson-Crick basepairing; ., non-canonical G. U interaction. (0) and (O), Residues that were strongly and weakly, respectively, modified by DMS, CMCT and RNase T,. A and A, Residues that were readily and less readily, respectively, cleaved by nuclease Vi. Shaded sequences are those that became reactive upon denaturation of the pre-rRNA molecule. The 6 substructures (I to VI) in this model are also indicated.

710

L.-C. C. Yeh and J. C. Lee

68 and 93 to 100, was either cleaved by the doublestrand-specific RNase V, or became accessible to chemical modifications following denaturation of the RNA molecule. At the foot of the stem are located two G. U pairs (G61. UlOO and G62. U99) followed by a G. C pair (C63. G98). Both G61. UlOO and G62. U99 pairs are non-canonical base pairs. G98 is located at the end of a stretch of purine residues adjacent to a pyrimidine. Residues C88, C89 and A90-92 were reactive with chemical probes weakly, even though these residues were seemingly involved in base-pairing according to the model. Furthermore, residues 76 to 86, located in the loop region in the model, should be accessible to singlestrand-specific probes. However, none of these residues was reactive in the native RNA and only became modified after denaturation of the RNA molecule. Reasons for this apparent anomaly are not clear. One possible explanation is that this region is not simply unstructured and is involved in interactions with a distal region of the 35 S prerRNA molecule. Possible interaction sites may be a stretch of unpaired adenine residues or a stretch of A. U pairs. Three stranded complexes of polyadenylic-polyuridylic acids have been shown to form (Felsenfeld et aE., 1957). (e) Substructure

I/

This region includes residues 103 to 187 arranged in a long helical structure with five small bulges and a small loop at the end. Most experimental results support the existence of this structure, i.e. residues at the bulges and the loop region (U112-A115, U144-148, A161-U163) were reactive readily whereas those in the helical regions were only accessible to single-stranded probes after denaturation of the RNA molecule. The proposed CZ cleavage site (Veldman et aZ., 1981) involving the sequence UCGUUU (residues 134 to 139) is located at the end of a relatively weak helical region with an estimated AG value of - 15 kcal. (f) Substructure VI This region consists of residues 188 to 211. Residues in the helical region (Al88 to Cl95 and G204 to U211) were either cleaved by RNase Vi or not accessible to chemical/enzymatic modifications unless the pre-rRNA was denatured. Such findings would suggest that these residues are base-paired. Residues Al79 to G201 were modified by singlestrand-specific probes. However, the adenine doublets (A202-203) were not accessible to the singlestrand probes, even though they were predicted to be located in a single-stranded loop region. It is tempting to speculate that several of the adenine and guanine residues in this region might interact with the stretch of uridine residues in substructure IV. Further experimentation will be necessary to verify or amend the proposed interactions. Earlier, a processing scheme had been proposed in

which all processing sites for the yeast pre-rRNA are clustered and that these cleavage sites are placed into close proximity by base-pairing between surrounding sequences (Veldman et ah, 1981). The processing sites are in a similar configuration consisting of a combination of a type I and a type II consensus sequence flanked by short base-paired regions. According to the present model for ITS-2, the previously proposed processing sites are located in short base-paired regions. Whether they are clustered as suggested cannot be clearly discerned by the present data. Secondary structure models for the ITS-2 region of S. carlsbergensis and of S. pombe were generated using the same computer program and similar assumptions. Models for S. carlsbergensis (Fig. 6(b)) and S. cerevisiae (Fig. 6(a)) are very similar. This is not surprising, since the ITS-2 sequences for the two yeasts are identical except for one residue. The variant nucleotide (U81 in S. cerevisiae is missing in S. carlsbergensis) is located in the U-rich loop in substructure IV. The model for S. pombe (Fig. 6(c)) is also similar to that proposed for S. cerevisiae, even though the S. pombe ITS-2 sequence is longer and has a lower G + C content than the S. cerevisiae sequence (Table 5). The extra nucleotides in S. pombe appear to be accommodated mostly in the long stem of substructure V. In both cases, the 3’ end of the 5.8 S rRNA sequence is hydrogen-bonded to the 5’ end of the 25 S rRNA sequence. A search in the S. pombe ITS-2 region for sequences that are similar to those proposed for the IS. cerevisiae processing sites fails to reveal their existence. Therefore, it is not clear that a consensus recognition sequence can be suggested. Perhaps, the recognition signals for the processing enzymes lie more in the higher-order structure than in the primary structure. Validity of the model will have to be tested with additional experimental data. Using the rDNA sequence data of Neurospora crassa (Chambers et al., 1986), mung bean (Schiebel & Hemleben, 1989) and X. laevis (Hall & Maden, 1980), secondary structure models for t,he ITS-2 region of these organisms were created (Fig. 6(d) to (f)). Although N. crassa is classified as a fungus, the model for this organism is distinctly different from those of S, pombe, S. cerevisiae and S. carlsbergensis. The N. crassa ITS-2 sequence is shorter than that of

Table 5 Size and G+C

content of internal transcribed 2 in precursor ribosomal RNAs

Organism 5. cerevisiae S. carlsbergensis S. pombe

N. cmaaa Mung bean x. lamis

spacer

No. of bases

G+C (Y*)

235 234 300 145 220 262

38.7 389 21.0 58.6 59.1 88.0

711

Yeast Precursor Ribosomal RNA Length:

(ai

443

Energy: -108.5

(b)

Length:

442

Cd)

Length:

353

Erie

S. cerevisioe

Energy: -102.3

s. pumbe

(e)

Figure 6. Schematic drawings of secondary structure models of the ITS-Z regions of several eukaryotes: (a) S. cerevisiae; (b) S. curbbergensis; (c) S. pombe; (d) N. crassa; (e) mung bean; and (f) X. laevis. **, The beginning of ITS-2 sequence; *, the beginning of the 25 S rRNA sequence. The ITS-2 sequence is shaded. Sequence data for the various ITS-Z regions are from (a) this laboratory, (b) Veldman et al. (1981), (c) Schaak et aE. (1982), (d) Chambers et al. (1986), (e) Schiebel & Hemleben (1989), and (f) Hall & Maden (1980). Length indicates the length of the entire sequence under consideration by the computer program in generating the secondary structure. Energy indicates the AG value (kcal/mol) for the proposed structure.

and has a higher G +C content S. cerevisiae (Table 5). Earlier, Chambers et al. (1986) compared the two sequences using a computer program of Wilbur & Lipman (1983) and found a lack of the ITS regions of homology in sequence 8. carlsbergensis and N. crassa. Models for mung

bean and X. laevis are surprisingly similar to that for S. cerevisiae, even though the ITS-2 sequences and the G +C content of these organisms are very different (Table 5). In conclusion, we have developed a structural model for the ITS-2 region of S. eerevisiae based on

712

L.-C. C. Yeh and J. C. Lee

theoretical free-energy calculation and experimental data. Theoretical models generated using identical assumptions for the ITS-2 region of S. pombe, S. carlsbergensis, mung bean and X. laevis have several common secondary structure motifs. Whether these common features of the helical structure involving the 3’ end of the 58 S and the 5’ end of the 25 S rRNA sequences and the cross-like ITS-2 structure play any role in the recognition mechanism of ribosomal RNA processing must await further experimentation. Nonetheless, the model for S. cerevisiae should provide a reasonable prototype for further study of the structure of the pre-rRNA molecule as well as interactions between pre-rRNA and ribosomal proteins during assembly. Support

from the

NIH

(GM35851)

is gratefully

acknowledged.

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