Accessibility of 5 S RNA in 50 S ribosomal subunits

J. XoZ. Biol. (1074) 90, 181-184

LETTERS TO THE EDITOR

Accessibility of 5 S RNA in 50 S Ribosomal Subunits Only two sites in d 8 RNA react with Kethoxe.1 in CiOS ribosornal subunits. Tl~cso two sites, G,, and G41, have previously beon found to bc accessible in free 5 S Rn’A. Kuclcotide sequences which have been suggested w possible bindirlz sites for the T-Y-C-G loop of tR.NA am not. accessible. The role of 5 S RNA in protein synt,hesis is not yet known. Its presence in the 50 S ribosomal subunit is required for several ribosomal functions (Erdmann ek al., 1971), but it is not clear whether it, is involved directly in any of these functions. Tn order to formulate more clearly experimental a,pproaches to t,his question, it would be helpful to know which sit.cs in this molecule are available for functional interactions with other ligands. Extensive work has been reported describing the accessibility of nucleotide rcsidues of 5 S RNA in solution to chemical modification and nuclcasc attack (Rrownlce & Sanger, 1969; Lee BEIngra.m, 1969; Jordan, 1971; Vigne & Jordan, 1971; Bcllemare et al., 1972a,b; Mirzabekov t Griffin, 1972). Oligonuclcotida binding st.udies have been used as a probe for tho regions of 5 S RNA which might bc available foi base pairing (Lea-is & Doty, 1970). It. is of great interest to know what relation these results have to the conformation of 5 S RX-A within the intact, functional ribosome. There is some disagreement about the conformation of 5 S RNA deduced by workers in different laboratories, possibly due to struct,ural rearrangement of t,hc molecule on exposure to various non-ribosomal cnvir0nment.s. In addition, assembly of the 50 S subunit may induce extensive masking or unmasking of regions of the polynucleotide chain. Thus, it. is important to examine the accessibility of 5 S RNA in the intact subunit. Tt has been reported t.hat 5 S RSA is inaccessible t.o nucleases in partially reconst.ituted and native 80 S particles (Reynier & Forget: 1970; Peunteun BEMonier, 1971). WC report here the ident.ification of two sites within the 5 S RXA in int,act 50 S subunits that are reactive toward the guanine-specific reagent Ket,hoxal. Ribosomes werp prepa.red from 32P-labeled Escherichia mli 3lRE690 after freczcthaw lysis in the presence o f lysozyme (Ron el al., 1966) and ccntrifugat.ion through a sucrose cushion in 05 nr-KH,Cl (Staehrlin et al., 1969). 50 S subunits, prepared as previously described (Noller et al., 1971). wcrc incubated at. a concernration of 1.25 mg/ml in a reaction mixt.ure containing 3 mg Kethoxal/ml (Nutritional Riochcmicals). 0.1 M-potassium cacod.ylate (pH 7.0) and 20 mal-MgC12z at 37°C for t,wo hours. dftcr precipitat.ionof 50 S subunit.s by addition of O@ivolumeof ethanol, RNAwasprel;arcd by three successive extractions with phenol (Traub et al., 1971)> carried out in the presence of 25 mhr-potassium borate. 6 S RNA was isolated by elcct,rophoresis on a 5~6 polyacrylamide slab gel (0.2 cm x 10 cm x 15 cm) made according to thcl method 01 Adams et aZ. (1969) and run for approximately three hours at IO V/cm at 6°C in 0.1 al-Tris-acetate. 0.02 >r-boric acid, pH 8.3. The 32P-labeled 5 S RSA band \vas located by autoradiography, elut.ed with two successive extractions of the materated gel slice by shaking wit.h O-5 ml 0.2 nr-sodium acetate (pH 5.0) for about one hour at WC. and recovered hy several precipitations with two volumcha of ethanol. 12

IS1

182

H.

F. XOLLER

ASD

W. HERR

Sites of reaction with Kethoxal were revca,led by a “diagonal” paper electrophoresis procedure, analogous to the met,hod described by Brown & Hartley (1966) for location of disulfide bridges in proteins. The 32P-labeled 5 S RN-4 is digested wit.h ribonuclease T, and alkaline phosphatase and subjected to one-dimensional electrophoresis on DEAE paper. After removal of Kethoxal from the blocked nucleotides, and digestion by an additional trea.tment with ribonuclease T, on the paper st,rip, a. second electrophoresis step is carried out, at right angles t.o the original direction. Nucleotides unaltered by the second step form a diagonal line and oligonucleotides originating from the site of Kethoxal attachment lie off the diagonal (Plate I(a)). The sites of attachment of Kethoxal can be unambiguously identified as any phosphorylated 3’-terminal guanosine residue in oligonucleotides lying off t.he diagonal. Pairs of nucleotides lying on a vert,ical line are adjacent in the RNA chain, the dephosphorylated one occupying the 3’ position relative to the phosphorylated nucleotide. Six off-diagonal spot.s are resolved by diagonal electrophoresis of 5 S RNA from Kethoxal-treated 50 S subunit8 (Plate T). The nucteotide sequences of the fragments TABLE

1

Sequences of donuckme Sucleotide

1 2 3 4 5 6

RNAase A products TJ, AGo” U, AGO,, C, G C, AC (1 AU,.4C,C,G

T, digomc1eotide.s

Deduced sequence U-.4X& U-A-G,,, CC-G p C-A-G, C-C-Go,, A-C-C-C-C-A-U-G

Nucleotide number?

0.35 0.14 0.32 0.13 0.95 1.00

t10 t.10 t6

p

Relative molar yield

$ t5 t,14

t According to the numbering system of Brownlee et nl. (1965). $ This oligonucleotide was not found by Brownlee et nl. (1968) but has more recently identified as a variant of nucleotide t5 in some strains of E. cdi (.Jarry & Rosset, 1971).

‘FABLE

?,

Sires of attachrnmd of h’ethoxal Kct.hoxal attachment site:

Oliponucleotitle pair

4-4

K C’-(‘-G-U-.4-G K C-A-(:-I’-A-C:

(:I:,

6--S

K A.CI.(.‘-C-(‘.A.l:-(:.(‘-(“.C.

G II

3 1

Q L3

t Sites of attnchmcnt of Kethoxal am indicated by the letter K. f According to the numbering system of Brownltvl et ~11.(1968).

been

$ z c i 9

Plate I. Diagonal electrophorosis of Kethoxal-treated 5 S RNA. Kethoxal-treated 32P-labolod 5 S 1 h with a mixture of RNAase T, and alkalino phosphatase as described by Barrel1 (1971), except Electrophoresis in the first dimension was on Whatman DE81 DEAE paper in 7% formic acid for 20 washed with 150 ml 0.013 M-Tris.base and incubated for 2 h at 37”C, then wetted with a solution of again incubated for 2 h at 37”C, and sown on to a second DEAli: shoot. Electrophoresis in the second

CCGoH

0 4

UAGoH

3 CCG,

0

’

0

RNA (4 x IO6 cts/ min, 5 pg) was digested at 37°C for for the inclusion of 0.1 mu-Z&l, and 20 mx-borate. h at 10 V/ cm. The &rip containing the nucleotides was Sankyo RNAase T, (0.05 mg/ml, 0.01 *I-‘l’ris, pI3 7.8), dimension was in 7% formic acid at 10 V/cm for 12 h.

5

0

CA%

UAG,,@

LETTERS

TO THE

EDITOR

183

determined by comparison of their ribonuclease A digestion products with the sequence of Brownlee et a2. (1967), are shown in Table 1. The six oligonucbotides pair with each other to form three sequences, as shown in Table 2. Reaction of Kethoxal is at the indicated guanine sites, which correspond to G,, and G,, in the 5 S RNA chain (Brownlee et al., 1967). Alignment of the oligonucleotide pairs is consistent with their position after diagonal electrophoresis, their relative molar yields (Table 1) and the known 5 S RNA sequence. These findings are in agreement with the results of Bellemare et al. (19723) who examined the reactivity of Kethoxal with 5 S RNA in solution, as well as with results of other chemical modification and nuclease susceptibility studies (Brownlee & Sa,nger, 1968; Lee & Ingram, 1969; Jordan, 1971; Bellemare et al., 1972a). None of the Kethoxal-reactive guaniue residues are shielded, and no new guanines exposed when the 5 S RNA is in its native conformation within the 50 S subunit. This is in contrast to the results of Reynier $ Forget (1976) and Feunteun & Monier (1971), who reported that 5 S RNA in partially reconstituted and native 50 S particles was strongly protected from nucleases. This may be attributable to inaccessibility of m&eases to sites easily penetrated by Kethoxal. Our results are consistent, however, with the structure proposed for a model complex of 5 S RNA, 23 S RNA and ribosomal proteins L6, LlS and L25 by Gray et al. (1973). Accessibility of 5 S RNA to Kethoxal has also been reported by Delihas et al. (1973). Some of the conformational models based on the solution properties of 5 S RNA (Cantor, 1967; Boedtker & Kelling, 1967; Raacke, 1967; Mirzabekov Q Griffin, 1972) are not consistent with our findings and thus do not reflect the conformation of the molecule within the ribosome. The secondary structure proposed by Brownlee et al. (1967) is consistent with the availability of sites G,, and GJ1, but does not account for the high degree of base pairing revealed by physical methods. In view of the specific interaction of 5 S RNA with 23 S RNA and several ribosomal proteins (Gray & Monier, 1971), it is likely that the “native ” conformation of 5 S RNA will be inextricable from the conformation of other ribosomal macromolecules. It has been proposed that 5 S RNA may participate directly in the binding of tRNA by the complementary binding of the G-T-Y-C-G sequence of tRNA to the G-A-A-C sequences around site G4* or G,,, (Forget & Weissman, 1967; Ofengand & Henes, 1969; Jordan, 1971; Dube, 1973; Erdmann et d., 1973). The inaccessibility of a small molecule such as Kethoxal to residues G,, and G1,,7 indicates that, if the latter hypothesis is correct, 5 S RNA or some neighboring region of the 50 S particle must undergo a conformational change for binding of tRNA to occur. It has already been pointed out that the T-Y-C-G sequence in tRNA itself is also inaccessible (Yoshida et al., 1968). It is interesting that nucleotides originating from Kethoxal reaction at G1, are found in a molar yield of 0.5 compared to those originating at G,, (Table 1). Brownlee et al. (1968) reported that, in half the 5 S molecules of E. coli strain MRE600, U is found in place of G at position 13. This suggests that the sites at positions 13 and 41 are equally accessible, but that the presence of U at position 13 in half of the molecules gives rise to a lower yield of this Kethoxal-reactive site. If true, this suggests that the nature of the nucleotide at position 13 is not functionally critical, although it is exposed to the ribosome, since some strains of E. coli have only G at this position (Brownlee et al., 1968). In addition, it has been reported that ribosomes reconstituted from 5 S RNA which has been formylated at position 41 are active

184

H.

Y. SOLLEK

AND

W. HERR

~TLvitro (Fahnestock & Komura, discussed by Bcllemare et al., 197%). These obscrvat,ions suggest that the only two guanine residues of 5 S RNA which are exposed in the ribosome may not be involved in ribosomc funct,ion. This work wus supporbd by U.S. Public Health Xntional Institute of Gcncra.1 Medical Sciences. l’llimann Laboratories LTniversit\: of California Santa C&, Cttlif. 95064, Received

3 Juno

Service

Grant

no. 17129-01

from

tllc

C.S.A.

1974

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