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Chloroplast Ribosome Structure ELECTRON MICROSCOPY OF RIBOSOMAL SUBUNITS AND LOCALIZATION OF N6, N6- DIMETHYLADENOSINE BY IMMUNOELECTRONMICROSCOPY *

(Received for publication, April 2, 1981)

Mona R. Trempe and Dohn G. Glitz From the Department of Biological Chemism, University of California-Los Angeles School of Medicine, Los Angeles, California 90024

Ribosomal subunits from the chloroplasts of Alaskan peas have been studied by immunoelectronmicroscopy. Electron micrographs of negatively stained small and large ribosomal subunits show particles of similar size and in the same characteristic projections described for the ribosomal subunits of Escherichia coli (Lake, J. A. (1976) J. MOL BioL 105, 131-159), although minor structural differences are apparent. High pressure liquid chromatographic analysis shows the modified nucleoside iV‘N-dimethyladenosine is conserved in chloroplast 16 S ribosomal RNA, presumably as two successive residues near the 3’ end. Antibodies directed against flN-dimethyladenosine were allowed to react with chloroplast 30 S ribosomal subunits. Electron microscopy showed individual subunit-antibody complexes and pairs of ribosomal subunits cross-linked by a single antibody. In 94% of the complexes observed, antibody contact was consistent with a dimethyladenosine localization near the end of the small subunit platform, in an area of subunit contact in the 70 S ribosome. This localization is analogous to the placement of Wfl-dimethyladenosine in the E. coli ribosome (Politz, S. M., and Glitz, D. G. (1977) Proc. NatL Acad S C ~ . U. S. A. 74,1468-1472).

Chloroplasts of green plants synthesize proteins on 70 S ribosomes. These chloroplast ribosomes are similar to those of prokaryotes, but they differ markedly from the 80 S ribosomes that are present in the plant cell cytoplasm. Bacterial and chloroplast ribosomes are similar in size, contain similar RNA components (23 S , 16 S, and 5 S), and have related mechanisms of translation and responses to antibiotics (for reviews, see Refs. 1 and 2). There is strong homology between the sequence of Escherichia coli 16 S ribosomal RNA and the DNA sequence coding for the 16 S ribosomal RNA of Zea mays (3). These facts lend support to the theory that chloroplasts are descended from prokaryotes (particularly cyano- bacteria) that became symbiotically associated with eukaryotic cells (4).

With regard to the E. coli ribosome, much is known about its overall structure and the interrelationships of several structural and functional components, particularly in the 30 S or small ribosomal subunit. A major technique used in these studies has been immunoelectronmicroscopy, the visualization in electron micrographs of antibody-linked biological struc-

* This work was supported by National Science Foundation Grants PCM 77-14872 and PCM 80-16598. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accord- ance with 18 U.S.C. Section 1734 solely to indicate this fact.

tures. It has been used to map the distribution of ribosomal proteins (5, 6), to localize the ribosomal neighborhood involved in puromycin binding ( 7 ) , and to investigate the structure of ribosomal RNA (8-10).

In contrast, rather little is known about the detailed structure of chloroplast ribosomes. The value of immunoelectronmicroscopy in studies of bacterial and rat liver cytoplasmic ribosomes (11) indicated that this technique could be of use in the study of chloroplast ribosomes. In this paper we describe the overall topography of both 30 S and 50 S ribosomal subunits from the chloroplasts of Alaskan pea seedlings and compare the structures with those of E. coli. In addition, as a specific probe of the ribosomal RNA, the location of the highly conserved residues of N6,N6-dimethyladenosine in the 30 S subunit has been demonstrated by immunoelectronmicros- COPY.

MATERIALS AND METHODS

Chloroplast and Ribosome Preparation-Chloroplasta were isolated from approximately 150 g of 12- to 14-day-old Alaskan pea seedlings by a method adapted from Mache et al. (12). The seedlings were kept for 16 h in the dark, washed briefly in cold 0.1% sodium hypochlorite, and homogenized for 15 s at 4 ‘C in a Waring Blendor using 3 ml of buffer (0.4 M mannitol, 50 mM KCI, 5 mM Mg-acetate, 6 mM 2-mercaptoethanol, 40 mM Tricine,’ pH 7.6)/g of tissue. After filtration through three layers of Miracloth (Chicopee Mills, Milltown, NJ) the chloroplasts were collected by centrifugation for 15 min at 3000 X g (5000 rpm in a Sorvall SS-34 rotor).

The chloroplasts were osmotically disrupted by adding 1 ml of lysis buffer (10 n“ Mg-acetate, 6 mM 2-mercaptoethanol, 40 mM Tricine, pH 7.6)/10 ml original volume. The suspension was stirred in the cold for 1 h and then centrifuged for 20 min at 27,000 X g (15,000 rprn in a Sorvall SS-34 rotor). Ten Fg of deoxyribonuclease I (Worthington Biochemicals, electrophoretically purified and ribonuclease-free) were then added to the supernatant.

The preparation of chloroplast ribosomes was based on procedures used with E. coli as described by Traub and Nomura (13). The solution above was layered over 6-ml pads of I M sucrose (Sigma, ribonuclease-free) in high salt buffer (300 mM NH4C1, 10 mM Mg- acetate, 6 mM 2-mercaptoethanol, 40 mM Tricine, pH 7.6) and centrifuged for 3 h at 200,OOO X g (55,000 rprn in a Beckman 60 Ti rotor). The resulting ribosome pellets were stored at -70°C until used.

Ribosomal Subunit Preparation-Fifty to 75 A260 units (3-4.5 mg) of ribosomes in 3 ml of dissociation buffer (50 mM NH4C1, 0.5 mM Mg- acetate, 10 mM Tricine, pH 7.6) were layered on three 56 ml 10-30% (w/v) sucrose gradients in the same buffer and centrifuged for 16 h at 23,000 rpm in a Beckman SW 25.2 rotor. The 30 S and 50 S subunits were pelleted by centrifugation of the appropriate pooled fractions for 3 h a t 200,000 X g (55,000 rpm in a Beckman Type 65 rotor). Subunits were either stored as pellets a t -70 “C or resuspended in ribosome buffer (150 XIIM NH4C1, 2 mM Mg-acetate, 10 mM Tricine, pH 7.6). Unless otherwise stated, all ribosome studies were carried out in this buffer. Typically, 150 g of leaves yielded 0.7-1.0 mg of 30

’ The abbreviations used are: Tricine, N-[Tris(hydroxymethyl)- methyllglycine; m!A, N6,N6-dimethyladenosine.

11873

11874

A Chloroplast Ribosome Structure

B

H FIG. 1. Electron micrographs of chloroplast ribosomal subunits. A, 30 S subunits; B, 50 S subunits. Bar length, IO00 A.

S subunits and 2-2.7 mg of 50 S subunits. Polyuridylic acid stimulated binding of [14C]phenylalanyl-tRNA"h"

was measured as described by Zamir et al. (14). ["CJphenylalanyl- tRNA"h" was purchased from New England Nuclear, and polyuridylic acid was from Calbiochem.

Quantitation of N",N"-Dimethyladenosine in 16 S Ribosomal RNA-RNA was isolated from approximately 0.5 mg of purified 30 S subunits by phenol extraction (13). The RNA was hydrolyzed to nucleosides with snake venom phosphodiesterase, bacterial alkaline phosphatase (Worthington), bovine pancreatic ribonuclease, and ribonuclease TI (Sigma) (15). The resulting nucleoside mixture was analyzed using reversed-phase high pressure liquid chromatography on a Waters Associates Clr-pBondapak (4 X 300 cm) column. The chromatographic system was modified from Gehrke et al. (16); the initial buffer included 99% of 0.01 M KHd'O., (pH 5.07) and 1% methanol. Nucleosides were eluted with a hyperbolic gradient to 50% methanol (Waters Associates curve 8) over 50 min, a t a flow rate of 1.5 ml/min. N",N"-dimethyladenosine was eluted a t 49 min. Purified nucleoside standards were purchased from Sigma.

Antibody Preparation and Characterization-The methods used to prepare the N".N"-dimethyladenosine bovine serum albumin con- jugate (17) and for immunization, blood collection and antibody characterization have been described (18). Antibodies were purified

from serum by precipitation from 40% saturated (NH,)YSO.~ solution, followed by passage through a column of DEAF,-cellulose overlaid with CM-cellulose (19). Proteins in the mixture were analyzed by polyacrylamide gel electrophoresis at pH 8.9 in 7.5% gels (20). All immunoglobulins used in these experiments were from the serum of a single rabbit (No. 57).

Electron Microscopy-Samples for microscopy were adsorbed to thin carbon films and negatively stained with 1% uranyl acetate using the double carbon technique developed by Valentine et al. (21) as modified by Lake and Kahan (22). Electron micrographs were obtained with a JEM l00B microscope a t 80 kV and an absolute magnification of 68,700. Free ribosomal subunits were used a t a concentration of approximately 0.05 mg/ml of ribosome buffer. In order to form subunit-antibody complexes, 40-50 pmol of 30 S subunits were incubated overnight in the cold with two m2A binding equivalents of anti-m;A antibodies in a total volume of 40-80 1.11 of ribosome buffer. Unreacted immunoglobulins were removed by Seph- arose 6B chromatography (8, 23).

RESULTS

Ribosome Preparations-Chloroplast ribosomes and ribosomal subunits prepared as described here are free of meas-

FIG. 2. Electron micrographs of chloroplast 30 S ribosomal subunits. Subunits are shown in the intermediate (50") view (Row A ) , the asymmetric (110") view (Row B ) and the quasisymmetric (0") view (Row C) as described by Lake (24) for E. coli 30 S ribosomal subunits. Below each frame is an interpretive drawing. Bar length, IO00 A.

FIG. 3. Electron micrographs of chloroplast 50 S ribosomal subunits. Subunits are shown in the quasisymmetric views (Rows A and B ) and the symmetric view (Row C) as described by Lake (24) for E . coli 50 S ribosomal subunits. Below each frame is an interpretive drawing. Bar length, 1O00 A.

Chloroplast Ribosome Structure 11875

A

B

A

0 0 . ... - . .,. . . - I

0 0 0 Q

C

H FIG. 2.

,- , . ._:

0

B

cs 0 ci"

C

c3 c3 c3 M

FIG. 3.

11876 Chloroplast Ribosome Structure

urable contamination by cytoplasmic ribosomes or ribosomal subunits, as measured by the polyuridylic acid stimulated subunits, as judged by sedimentation profides and electron binding of [‘4C]phenylalanyl-tRNA”h’, was equivalent to that microscopy. Only 70 S particles or 30 S and 50 S subunits we observed in functional E. coli 30 S ribosomal subunits. were observed in preparative sucrose gradients, and images Electron Microscopy of Chloroplast Ribosomal Subunits- characteristic of the larger cytoplasmic subunits were never Purified subunits from chloroplasts were negatively con- seen in electron micrographs. The activity of chloroplast 30 S trasted and viewed in the electron microscope. Typical fields

of 30 S subunits (Fig. lA) and of 50 S subunits (Fig. 1B) show structures that strongly resemble the corresponding structures seen in micrographs of E. coli ribosomal subunits stained with the same double-carbon technique. The maximum dimension measured in micrographs of the chloroplast 30 S subunit, 235 k 15 A, is similar to the 250 f 30 8, reported by Lake (24) for the E. coli 30 S subunit. The maximum dimension of the chloroplast 50 S subunit, 280 f 10 A, is also similar to the 275 f 15 A of the E. coli 50 S subunit (24).

The characteristic projections of the E. coli ribosomal subunits as described by Lake (24) can be identified in the chloroplast small and large subunits. Fig. 2 presents a gallery of chloroplast 30 S subunits in these characteristic views; each asymmetric projection is shown in both possible orientations. The intermediate (50’) view (Row A) is characterized by a clearly defined lump or platform. The asymmetric (1 10’) view

FIG. 4. Quantitation of pfl-dimethyladenosine in an enzymatic hydrolysate of chloroplast 16 S ribosomal RNA by

IO 20 x) 40 30 YI*UTLS reversed-phase high pressure liquid chromatography

A B .~ .,...”,* T”.,””r\y ._.. ~ , - . . “r : . z \~r . ,~ l“ . .. - ~ .- .-,-. .. -..>,. . , .. I..

FIG. 5. Electron micrographs of chloroplast 30 S ribosomal subunits complexed with anti-Mfl-dimethyladenosine immunoglobulins. A, subunit-antibody monomers; B, antibody-cross-linked subunit dimers. Complexes are indicated by arrows. Bar length, IO00 A.


(Row B) is characterized by one convex side and one concave side, but the platform is not clearly separated from the body of the subunit. Row C shows the quasisymmetric (0') view, characterized by a line of approximate mirror symmetry. Although the chloroplast small subunit clearly exhibits the same characteristic views as the E. coli 30 S subunit, the two structures do not appear entirely identical. In the chloroplast small ribosomal subunits, the head or upper one-third region appears slightly smaller than we see in the E. coli 30 S subunit, the platform structure appears blunter and more defined, and a clear line of stain is more often seen along the division between the upper one-third and lower two-thirds of the chloroplast subunit.

Fig. 3 presents a gallery of chloroplast 50 S subunits. Rows A and B show two variants of the Lake (24) quasisymmetric view, as characterized by a central protuberance at the top of the subunit and a rod-like appendage identified as ribosomal protein L7/L12 in E. coli (25). Depending on the orientation of the ribosomal subunit with respect to the supporting carbon film, this arm is either aligned radially (Row A) or extends roughly horizontally from the subunit (Row B). Row C shows the asymmetric view, characterized by a convex lower edge and a cleft in the rather flat upper boundary. Each view of the 50 S subunit is shown in both enantiomorphic orientations. Again, although very similar, there are slight differences between the chloroplast 50 S subunit and that of E. coli as judged from electron micrographs. In the quasisymmetric view, the rod-like arm appears slightly longer and a small indentation is more often seen at the lower edge of the chloroplast subunit.

Quantitation of N6,N6-Dimethyladenosine in the RNA of

r* , .. . "

B

C

. . , I .

e,

the Chloroplast Small Ribosomal Subunit-Chloroplast 16 S ribosomal RNA was enzymatically hydrolyzed to nucleosides. Fig. 4 shows the high pressure liquid chromatography trace obtained from 0.2 nmol of hydrolyzed RNA. The N",N"-dimethyladenosine peak is well separated from all other nucleosides present in the digest and gives a value of 1.5 mol of mgA/mol of RNA. An identical hydrolysate of E. coli 16 S ribosomal RNA gave a value of 1.3 mol of m2A /mol of RNA. Each result is somewhat lower than the expected value of 2 mol of mgA/mol of RNA, perhaps due to the resistance of the m;A-m;A dinucleotide to enzymatic as well as chemical cleav- age (26).

Electron Microscopy ofAntibody-Ribosomal Subunit Com- plexes-Anti-mzA antibodies were incubated with chloroplast 30 S subunits. Unreacted immunoglobulins were removed by gel fitration and the antibody-ribosomal subunit complexes were negatively contrasted. Electron microscopy showed two types of complex: 1) monomers, in which only one antigen- binding site of an IgG molecule had reacted with the m;A in the 30 S ribosomal subunit; and 2) dimers in which both antigen binding sites of an IgG molecule were filled, resulting in an antibody bridge between two ribosomal subunits. Fig. 5 shows fields of each type of complex: panel A illustrates monomer structures and panel B shows antibody-linked dimers.

The location of the N",N6-dimethyladenosine residues in the 30 S chloroplast ribosomal subunit was approximated by analysis of the apparent point of contact of antibody to the ribosomal subunit in each of the characteristic orientations. About 100 micrographs showing approximately 6000 small subunits were examined, and 495 antibody-subunit interac-

;...-77r,. .. .,

c9

Ga

W tp

03 cp

co 90 V H

FIG. 6. Electron micrographs of chloroplast 30 S ribosomal subunit-antibody complexes. Subunits are shown in the intermediate ( 5 0 " ) view (Row A ) , the asymmetric (110') view (Row B ) , and the quasisymmetric (0") view (Row C) as described by Lake (24) for E. coli 30 S ribosomal subunits. Below each frame is an interpretive drawing. Bar length, IO00 A.

11878 Chloroplast Ribosome Structure

A

QQ

- FIG. 7. Electron micrographs of antibody-linked subunit dimers. Below each frame is an interpretive drawing. Bar length, lo00 A.

d 50' I lo'

0' so* I10'

FIG. 8. Comparison of the localization of NBw-dimethyladenosine in 30 S ribosomal subunits. The shaded areas indicate the binding site of anti-m?A antibodies to the E. coli 30s ribosomal subunit (top TOW from Ref. 8 ) and to the chloroplast 30 S ribosomal subunit (bottom row). Subunits are drawn in the quasisymmetric ( O " ) , intermediate (50"). and asymmetric ( 1 10") views as described by Lake (24).

tions were clearly identified and evaluated. All except 30 (6%) of these images were consistent with the localization described below.

Fig. 6 shows a gallery of antibody-subunit monomers ar- ranged in the characteristic subunit projections. In the intermediate (50') view (Row A), the antibody molecules are consistently seen attached to the platform, at or near the end of the structure, regardless of the orientation of the subunit. In either orientation of the asymmetric (110') view (Row B), contact with antibody is a t the convex side of the structure. In the quasisymmetric (0') view (Row C) the antibody is usually partially obscured, and the site of antibody attachment appears slightly below the partition between the upper and

lower parts of the subunit. The visible portion of the antibody molecule can lie either to the left (first 3 frames of Row C) or to the right (last 3 frames) of the subunit.

Fig. 7 shows a gallery of antibody-linked subunit dimers in which each of the views described above can be seen. Again, the site of antibody attachment is to the convex side in the asymmetric view (see Row A, first and third frames; Row B, 5th frame) or to the platform structure in the intermediate view (see Row B, 2nd frame).

The position of antibody attachment observed in both monomers and in antibody-linked dimers is consistent with a single binding site which localizes the m$A residues near the end of the platform structure, as shown in Fig. 8 (bottom row). If compared with the placement of them$A residues in the E. coli 30 S ribosomal subunit (Fig. 8, top row) (8), the localization in the chloroplast 30 S ribosomal subunit appears somewhat closer to the end of the platform but in an otherwise analogous position. Hence, the close similarity in structure of prokaryotic and chloroplast ribosomal subunits extends also to a nearly identical placement of the widely conserved N",N"- dimethyladenosine residues.

DISCUSSION

The results presented in this paper are fully compatible with the hypothesis that chloroplast ribosomes are closely related to and perhaps descended from prokaryotic ribosomes. In electron micrographs, negatively contrasted ribosomal subunits from chloroplasts appear very similar in size and present the same characteristic projections as the corresponding subunits from E. coli. Only minor differences in topography are seen. This structural similarity is further emphasized by the mapping of a specific portion of the 16 S ribosomal RNA in the 30 S subunit. The localization of N",N"-dimethyladenosine in the chloroplast 30 S subunit is completely analogous to the localization in the E. coli 30 S subunit.

Our localization of the N",N"-dimethyladenosine residues places them on the platform of the small subunit. The platform occurs at the subunit interface when E. coli small and


large subunits combine to form an active 70 S ribosome (24). Since the chloroplast subunits are so similar to those of E. coli and since micrographs of Euglena and spinach chloroplast 70 S ribosomes (27) show a similar juxtaposition of subunits, it appears safe to conclude that the N',N'-dimethyladenosine residues of the chloroplast 30 S subunit are similarly placed at or near the interface of the subunits in the 70 S particle.

The nucleotide sequence of the Zea mays chloroplast 16 S ribosomal RNA gene is known (3), but modified nucleosides cannot be unambiguously placed in this DNA sequence. How- ever, it seems almost certain that the N',N6-dimethyladenosine residues occur 24 and 25 nucleotides from the 3' terminus of the RNA. First, this segment of the chloroplast RNA sequence is strongly homologous with that of E. coli. Second, two successive N',N'-dimethyladenosine residues appear to be a universal feature near the 3' end of small subunit ribosomal RNA species. The sequence may be found in all eukaryotic cytoplasmic 18 S RNAs (28), and it is also present in an analogous position in hamster mitochondrial ribosomal RNA (29). Euglena chloroplast 16 S RNA has been reported to lack the characteristic N',N'"dimethyladenosine TI-oligonucleotide (30), but the ribonuclease TI-oligonucleotide which would be generated upon hydrolysis of the Zea mays RNA is only a tetramer (3) and would not have been detected by Zablen et al. (30).

In the attempt to define the role, if any, of the N',N'- dimethyladenosine residues in subunit function or interaction, much use has been made of comparison of wild type E. coli with a kasugamycin-resistant mutant that lacks the methylase responsible for this modification (31, 32). After investigation of the interaction of components involved in the initiation cycle, the only differences reported betwen mutant and wild type ribosomes were a slightly increased requirement for initiation factor 3 (33, 34) and a reduced level of ribosome- bound protein S1 in the mutant (35). Possibly more meaning- ful differences are seen when the effect of the methylated residues on RNA secondary structure is considered. The elec- trophoretic mobilities of RNA fragments containing the mtA residues are different from those of the analogous fragments from the mutant which do not contain the m$A residues. Thus, the change in stacking properties with methylation of the adenosines (36, 37) may have an effect on the local conformational stability of the ribosomal RNA (38). These apparently ubiquitous methylated residues may therefore act in subunit-subunit interaction, a necessary contact for protein biosynthesis in all cells, by controlling the conformation ofthe RNA at the site of interaction.

Acknowledgments-We thank Dr. J. P. Thornber and his associates for advice on chloroplast isolation and for assistance in obtaining fresh plant materials. We are also indebted for Dr. F. Eiserling and his colleagues for advice and for access to equipment for electron microscopy and photography.

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