J. Mol. Biol. (1990) 211, 907-918


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Tertiary Structure of 16 S rRNA Melanie I. Oakes, Lawrence Kahan and James A. Lake Molecular Biology Institute and Department of Biology University of California at Los Angeles Los Angeles, CA 90024, U.S.A. (Received 12 June 1989; accepted 19 September 1989) Seven regions of 16 S rRNA have been located on the surface of the 30 S ribosomal subunit by DNA-hybridization electron microscopy. This information has been incorporated into a model for the tertiary structure of 16 S rRNA, accounting for approximately 40% of the total 16 S rRNA. A structure labeled the platform ring is proposed for a region of rRNA within the central domain. This structure rings the edges of the platform and includes regions 655-751 and 769-810. Another region, the recognition complex, consists of nucleotides 500 to 545, and occupies a region on the exterior surface of the subunit near the elongation factor Tu binding site. Ribosomal proteins that have been mapped by immunoelectron microscopy are superimposed onto the model in order to examine possible regions of interaction. Good correlation between the model locations of ribosomal proteins, and regions of rRNA protected by ribosomal proteins provide independent support for this model.

1. Introduction

distribution of several regions of 16 S rRNA within the 30 S ribosomal subunit. The regions that have been localized as well as the portion of 16 S rRNA included in the model are demonstrated in Figure 1. Additionally, this model is compared with the experimental results and experiments of others. Data obtained by DNA-hybridization electron microscopy nicely complements the results of other techniques that are being used to determine the three-dimensional structure of rRNA. The model presented here is consistent with many of these results.

A variety of experimental approaches are contributing new information about the tertiary structure of ribosomal RNA. Models for the tertiary structure of 16 S rRNA have been proposed (ExpertBenzacon & Wollenzien, 1985; Schuler & Brimacombe, 1988; Stern et al., 1988c) based on data obtained from a variety of techniques including RNA-RNA crosslinking, RNA-protein crosslinking and ribosomal protein protection of rRNA. Two of these models also have incorporated neutron diffraction information on the placement of the ribosomal proteins (Cape1 et al., 1987). The aim of these models is to increase our understanding of the ribosome structure in order to determine the mechanisms of protein synthesis. DNA-hybridization electron microscopy is a useful technique to map single-stranded rRNA sites in three dimensions (Oakes et al., 1986). Thus far, seven 16 S rRNA sequences have been localized on the surface of the 30 S subunit. These sequences are: 16 S rRNA 518-533, 686-703, 714-733, 787-803, 1392-1407 and 1492-1505, and a Shine-Dalgarno sequence (Oakes et al., 1986, 1987; Oakes & Lake, 1990). Another region that has been localized by a variation of this technique is 16 S rRNA 1531-1542 (Olson et al., 1988). In this paper, we use these DNA-hybridization results t,o derive a model for the 0022-2836/90/040907-l

2 $03.00/O

2. Materials and Methods DNA probes were synthesized to complementary single-stranded regions of 16 S rRNA and DNA-hybridization microscopy was performed as described in the accompanying paper (Oakes t Lake, 1990). In the electron micrographs, the traditional projections (Lake, 1976) of the 30 S subunit can be identified as well as avidin, which is bound as a result of hybridization of the DNA probe. For each projection and each DNA probe, the region on the 30 S subunit surface that bound the probe was determined. Models of the tertiary rRNA st,ructure were constructed using cylinders and flexible connectors to represent helices and single-stranded regions, respectively. The 16 S secondary structure


model of Escherichia co2i 16 S rRNA 0 1990 Academic Press Limited


M. I. Oakes et al.

Figure 1. Secondary structure of 16 S rRNA in E. coli 30 S ribosomal subunits illustrating the regions mapped and the regions included in the 3-dimensional model. The underlined regions are the sequences that hybridized to complementary DNA probes. The hatched area shows regions excluded from the model. The secondary structure is adapted from Stern et al. (1988a,b,c).



Electron Microscopy

3. Model Building and Discussion Before starting in earnest to develop a detailed model, we consider how much rRNA can be packed

into a ribosomal subunit or into part of a subunit. Our assumptions are similar to those of Noller & Lake (1984). This depends in part on the dimensions of the RNA double helix. Helical RNA exists in a form whose structural parameters are closely related to the A form of DNA (Dickerson et al., 1982), with a helical repeat of approximately 11 base-pairs per turn. Axial spacing between basepairs



of 2.5, 2.6 and 2.8 A has been reported


Figure 2. Illustrations of the path at the 16 S 5’ terminus, of the pseudo-knotted helices, and of the 3’ regions of rRNA within the 30 S subunit forming a coaxially stacked helix. The small subunit is illustrated in the asymmetric view, as seen from the cytoplasm. (a) View the 5’ and pseudo-knotted regions showing (29-37/547-556). These structures are normally obscured by overlaying structures. (b) View showing the entire region.

was adapted from Gutell et al. (1986), with the following nucleotides and differences: M-566/884-886 888-891/909-912 were unpaired (Noller et al., 1987). The helices were scaled to the 1@5 base-pairs/helical turn with a rise of 2.7 A/base-pair and a helical diameter of 22 A (1 A = @l nm). We used 5 A for the average singlestranded nucleotide length. The overall dimensions of the model were scaled to those of a 30 S ribosomal subunit as determined by electron microscopy and X-ray scattering. Finally, for illustrative purposes, the helices are drawn in the RNA A conformation. Details of these are discussed in the model-building section. Placement of ribosomal proteins was based on immunoelectron microscopy (Lake, 1985; Oakes el al., 1987) and includes some of our unpublished results. Other information that was incorporated into the model was RNA-ribosomal protein interactions (Stern et al., 1986; Osswald et al., 1987; Greuer et al., 1987).


tRNA, A-DNA and RNA, respectively (Rich & RajBhandary, 1976; Jack et aE., 1976; Dickerson et aE., 1982). For this paper, we have used a figure of 2.7 A per base-pair and a diameter of 22 8. This corresponds to a distance of approximately 30 A per helical repeat (11 base-pairs). The second part of the model-building equation is the size of the ribosomal subunits. The best estimates from electron microscopy and low-angle X-ray scattering are 240 to 250 A for the longest dimension of the small subunit and 240 to 250 f! for the longest dimension of the large subunit (for discussion, see Lake, 1976). Thus, we have scaled our ribosome models to this size and assume they are accurate to within +/1Oo/o. A recognition


containing g-13/21-25, Helices sequences 17-19/916-918 and 27-371547-556 have been coaxially stacked. The coaxial stacking in this region is similar to that predicted in earlier tertiary models (Noller & Lake, 1984) and is based on stacking observed in tRNA for the acceptor and TI//C stem. This pseudoknot structure (Pleij et al., 1985) forms a bridge from 54 binding domains to 57 binding domains. These helices are shown in Figure 2(a), with nucleotides 37/546 positioned at the concave edge of the subunit. Constraints on the placement of these helices are the immunoelectron mapping of the 5’ end and reports that ribosomal proteins S4, S5 and S12 (Mochalova et al., 1982; Stern et al., 1986, 1988b) protect this region. When we place 37/546 at the edge of the subunit, the sequence from 501 to 545 consisting of two helical regions separated by an unpaired nucleotide could be placed either above (on the “cytoplasmic” surface) or below (on the “interface” surface) the pseudoknotted helix. A primary constraint on the model, illustrated in Figure 3(a), is that it places the sequence 518-533 within the region on the 30 S subunits where the same sequence was mapped by DNA-hybridization electron microscopy. Since our mapping indicates nucleotides within the 518-533 regions are located on the exterior or cytoplasmic side of the subunit, we favor placing this region above the pseudoknot. structure as indicated in Figure 2(b). This placement differs from that of Stern et al. (1988a,b,c), who have placed these helices on the “interface” side. Our placement is consistent with immunoelectron microscopic locali-

M. I. Oakes et al.

Figure 3. Comparison of the 16 S model with the sites mapped for rRNA sequences. Ribosomal RNA sites? as mapped, are shown by cross-hatching. Sites predicted from the model to lie within the cross-hatched region are shown on the 16 S molecule by thin lines. The last 2 nucleotides adjacent to the site labeled with biotin in the construction of the probe are shown by bold lines. (a) region 518-533; (b) region 139221407; (c) region 1492-1505; (d) Shine-Dalgarno region, 1534-1542; (e) region 787-803; (f) re g ion 686-703; and (g) region 714-733.

zations of ribosomal proteins S4, S5 and S12, and the modified nucleotide 7-methylguanosine G527 (Winklemann et al., 1982; Trempe et al., 1982). The location on the 30 S subunit for ribosomal protein 54, mapped by immunoelectron microscopy (Winklemann et al., 1982), has been superimposed on our model in Figure 4(a). This provides a test for the model. Since S4 is known to protect several 16 S rRNA regions from chemical modification during assembly, we have compared the overlap of the protein and rRNA mappings with the protection experiments of Stern et al. (1986). The outlined nucleotides that coincide with the protein mappings and hence could possibly interact with 54 are indicated by a continuous line on the rRNA secondary structure. Nucleotides that are protected by S4

during assembly are indicated by filled circles. This same representation has been used for ribosomal proteins 55 and S12 as well (Fig. 4(b) and (c), respectively). Some data suggest that 54, 55 and S12 form a functional complex. In particular, mutational alterations of both S4 and 55 are known to suppress the streptomycin-dependence phenotype of some S12 mutations (Anderson et al., 1967; Gorini, 1971) and these three proteins appear to co-operate in ribosomal control of translational fidelity (Gorini, 1969; Kuwano et al., 1969). Furthermore, the recognition binding site for elongation factor Tu has been mapped on this exterior surface of the small subunit (Langer & Lake, 1985; Girsovitch et al., 1986). The correspondence between our model and the results of protection studies is quite good.


Electron Microscopy

Fig. 3.



M. I. Oakes et al.

(b) The pZatform ring The central domain of rRNA extends from the pseudoknot and comprises the platform and other structures. Three sequences within the central domain were mapped on the platform by our DNAhybridization electron microscopy experiments. These are regions 686, 714 and 787. Since the platform is thin (approx. 30 A; Lake, 1976), it is unlikely to be more than a single RNA helix thick. This strongly constrains models for the distribution of rRNA on this region. Since region 787 is found at the top of the platform, we have placed the loop there. The 787 region is found at the top of the platform as shown in Figure 5(a), and the 686 region maps lower down on the platform on the convex surface. Likewise, the “elbow” at 714-733 that links helices on either side of it, has been placed on the convex lower surface of the platform. We have coaxially stacked helices 576-5871754-766 and 588-617/623-657 in the usual manner, since no unpaired nucleotides interrupt formation of the stacked helices. This allows more room for the lengthening of this region in small subunit eukaryotic rRNA. Figure 3(e), (f) and (g) demonstrates the relationship between the model of the central domain and the experimentally determined regions of 16 S rRNA mapped by DNA-hybridization electron microscopy. Other proposed models have included regions 655-751 and 768-810 in the platform and have placed the helices in various orientations (Stern et al., 1988c; Schuler & Brimacombe, 1988; Noller & Lake, 1984). The Schuler & Brimacombe

(1988) model describes

a parallelogram


four of the helices in the central domain. In the model proposed by Stern et al. (1988c), the central domain is described as a left shoulder-arm. Our model proposes that regions 655-751 and 768-810 are connected through their terminal loops into a ring structure. The name of this structure, the platform ring, conveys the general shape. Many platform ribosomal proteins are associated with the central domain of ribosomal rRNA. These include proteins S6, Sll, S18, S21 and, to a lesser extent, SB. Detailed information regarding interactions with specific nucleotides has been gained largely through crosslinking (for a review, see

Figure 4. Diagram of regions of contact between ribosomal proteins involved in recognition (S4, 55 and Sl2) and the 5’ region of 16 S rRNA. The filled dots indicate nucleotides protected from chemical probes by the respective proteins during assembly (Stern et al., 1988a,b,c). Cross-hatched areas are sites mapped for the respective proteins. The outlined regions of nucleotides coincide with sites where proteins have been mapped by immunoelectron microscopy. Pu’ucleotides 557 to 569 are not illustrated in the model structure. (a) The location of S4 determined by immunoelectron microscopy (Winklemann et al.. 1982). (b) The location of S5 determined by immunoelectron microscopy (Lake & Kahan. 1975). (c) The location of 812 determined by immunoelectron microscopy (Winklemann et al., 1982).


Electron Microscopy


Figure 5. Illustrations of the 3-dimensional structure within the central domain of 16 S rRNA. The subunit is shown in the asymmetric view, viewed from the cytoplasm. (a) Diagram of the platform ring, showing its connections. (b) Illustration of the connections of the central domain with the pseudoknotted helical region. (c) General schematic of the structure proposed for the entire central domain (helix 888-912 not shown).

Brimacombe et al., 1988) and protection from chemical probes (for a review, see Stern et al., 1988c). Figure 6 illustrates the locations of these proteins determined by immunoelectron microscopy. Overlap of this region with the 16 S rRNA is indicated on the side, as are the nucleotides that are protected by these respective proteins. Three of these immuno-protein mappings (515, 518 and S21) have not been published. Protection by the joint addition of proteins S6 and 518 is shown in Figure 6(a) and (b), since these studies were performed with both proteins present. Limitations of the protein assembly/protein studies are discussed elsewhere (Stern et al., 1988c). Our protein mappings are low-resolution mappings and, hence, the hatched area may include a larger area than the protein actually covers. Also, we have not

attempted to deduce whether a crossing strand of rRNA might protect a lower-lying strand. We simply have indicated all rRNA regions that lie within the cross-hatched areas and hence might be protected by protein binding. The number of nucleotides protected and not protected by the “platform proteins” is shown in Table 1. In general, the model is in good agreement with the results of the protein protection (73 of 84 nucleotides are accounted for). Other reported interactions that are accommodated by our model are the crosslink between the 690 and 790 regions (Atmadja et al., 1986), ribosomal protein crosslinks between Sl 1 and 693-697 and 702-705 (Greuer et al., 1987). The region from 811 to 879 has not been accessible to our DNA probes. Unlike the highly conserved regions within the platform, this region is


M. I. Oakes et al.

prdecm Experments are for 56 + SW


Figure 6. Diagrams illustrating regions of potential contact between ribosomal proteins S6, S8, Sll, S15, S18 and S21, and the central domain of 16 S rRNA. The filled dots adjacent to specific nucleotides indicate their protection from chemical probes during subunit assembly (Svensson et al., 1988; Stern et al., 1986). The outlined regions represent nucleotides in our model of 16 S rRNA that are covered by the sites mapped for the respective protein. (a) The locations of S6 determined by immunoelectron microscopy (Kahan et aZ., 1981), and the protection by the combination of proteins

DNA-hgybrid~izatim Electron Microscopy


86 and 818. (b) The location of 818 (our unpublished results) and t’he protection by the combination of protein &I and S18. (c) The location of 88 determined by immunoelectron microscopy (Kahan et al., 1981). (d) The location of X21 (our unpublished results). (e) The location of’ Al 1 determined hy immunoelertrnn microscopy (Lake & Kahan. 1975). (f) The location of 816 (our unpuhliahhed results).

M. I. Oakes et al.


Table 1 Nucleotides protected within cross-hatching

Protein Platform

Nucleotides protected and not in cross-hatching


Intermediate 58 Recognition

7 0 1 0

20 15 5 1

96fS18 Sll 915 94

between platform 14

and neck 0


s4 s5 s12

6 6 6

0 0 4




Nucleotides within the tertiary model of 16 S rRNA that are adjacent to ribosomal proteins (localized by immunoelectron microscopy) are compared with nucleotides protected by ribosomal protein footprinting (Stern et al., 1986, 1988a,b; Powers et al., 198&b; Svensson et al., 1988). The cross-hatching used to define nucleotide and protein overlaps are shown in Figs 4 and 6.

phylogenetically variable. It is largest in eukaryotes, intermediate in eocytes like Sulfolobus, and smallest. in eubacteria. Our placement of this helix is given slight additional support by computerof 30 S subunits from analyzed projections Saccharomyces cerevisiae, Sul’olobus acidocaldarius, Methanobrevibacter smithii and Escherichia coli. In preliminary correspondence analyses with J. Frank and A. Verschoor (for a projected structure, see Lake, 1988) the lobe of density that would correspond to the 840 loop and stem is largest in eukaryotes, intermediate in eocytes and smallest in eubacteria in accord with the observed phylogenetic variation of this region. For sequences 821-879, we have used the appealing model presented by Stern et al. (1988a,b,c), which is supported by crosslinking as well as phylogenetic evidence (Haselman et al., 1989) to intercalate helix 2 (as shown in Fig. 5(b)) between helix 1 and basepair 568-881. We have also oriented the terminal loop (841-845) towards the convex platform surface as shown in Figure 5(c); since G844 is protected by a combination of proteins 6 and 18. Placing this region below does not interfere with a tightly packed (when the dimethyl A helix is included) platform ring and allows it, to extend out of the platform so that its phylogenetic variations can be accommodated. Other interactions within the platform region that are compatible with the model include the crosslinking between S18 and 845-851 (Greuer et al., 1987) and a tertiary interaction between 570 and 866 (Gutell et al., 1986). Ribosomal protein S8 binds to the central domain of 16 S rRNA and occupies a region between the platform and the recognition proteins. Figure 6(c) illustrates the site mapped for S8 and the region of 16 S rRNA that could interact with 58. Again, nucleotides protected by 58 from chemical probes

are within the outlined regions (Stern et al., 1988c). Two (of 3) observed crosslinks of 651-654 and 629-633 to S8 are consistent with this placement (Wower & Brimacombe, 1983). We did not explicitly model the RNA in the region from nucleotide 884 to the pseudoknot structure that pairs nucleotides 916 to 918 with nucleotides 17 to 19. The region from 887 to 912 is, nevertheless, fairly well const’rained to the neck region of the subunit through its connections to neighboring helices. Within this region, numerous S5 and S12 protections are found. Notable are the S12-specific protections of nucleotides 886-917 (Stern et al., 1988b). Other associations of S12 with this region have come from antibiotic resistance studies of strepto1969; mycin (Ozaki et al., 1969; Birge & Kurland, Montandon et al., 1985). Likewise, we have not, explicitly modeled the path of the rRNA beyond t’he 923-933/1384-l 393 helix,where the RNA leaves the neck and enters into the head (Fig. 2(b)). The placement of this helix in the head is strongly supported by the mapping of the 1400 region of rRNA on the neck of the subunit near the platform in both E. coli and Saccharomyces (Oakes et al., 1986). Protein S7 binds t,o the part of the rRNA within the head of the 30 S subunit (for a review, see Noller & Woese; 1981). Protein S7 (located on the back of the head. on the interface side (Lake, 1985)) protects nucleotides 935 to 945 (Powers et al., 1988a) and a S7 crosslink has been reported close to this region at 1378 (Wower & Brimacombe. 1983). The penultimate and the dimethyladenosine helices are the last two to be positioned. Three regions within the 3’.terminal end have been placed on the surface of the 30 S ribosomal subunit. The relationship between the model for this region and the mappings are demonstrated in Figure 3(b), (c) and ((1). The penultimate helix (1409-1492) has been referred to as the cleft anchor (Noller & Lake, 1984) although its exact orientation is unknown. It is flanked at the 5’ and 3’ ends by two highly conserved regions. The 3’ flanking region (mapped in the accompanying paper) enters into the platform, while the 5’ flanking end emerges from the subunit in the vicinity of the cleft (Oakes et al.. 1986). Contained within these specific regions are the colicin E3 scission site (Bowman et al., 1971; Seniro & Holland, 1974), a site protected by IF3 (Wickstrom, 1983) and the site of crosslinking of Cl400 to the anticodon of a I’ site tRNA (Prince et al., 1982). The penultimate helix itself exhibits marked phylogenetic variability in length (Woese et al., 1983). Although our data do not significantly constrain the placement of the penultimate helix, its location near the base of the subunit will position the eukaryotic expansion segment at the loop of the helix in the most structurally variable part of the small subunit. This is, of course, appropriate for a phylogenetically variable feature for this region. The top of the helix is constrained to the cleft at one end but there are few restrictions on the lateral positioning of the stem, particularly toward the bottom of the subunit. At 1492. the rRNA leads


back into the platform where we have mapped sequence 1506-1529. Additional evidence for placing the dimethyl-A helix 1506-1529 on the platform comes from immunoelectron microscopy of the dimethyladenosine nucleotides and of the 3’ end (Politz & Glitz, 1977; Evstafieva et aE., 1983; Trempe & Glitz, 1981). The dimethyl-A helix could be placed in the center of the platform ring in either of two orientations. We have positioned dimethyladenosine nucleotides toward the bottom of the platform but this helix could also be placed in the opposite orientation with the dimethyladenosine nucleotides pointing towards the top of the platform. In either orientation, the Shine-Dalgarno region could still be accessible for binding to the mRNA upstream from the start codon. DNA-hybridization electron microscopy enabled the localizations of seven regions on the surface of the 30 S subunit. The majority of the regions are loops that are present at the ends of helices. Thus, our mappings constrain one end, or both ends of the helices. Starting from the 5’ end, we have described how our experimental results suggest that the 16 S rRXA may be folded. The majority of the 5’ domain is excluded from the model, since sequences in these domains have not been localized by DNA-hybridization microscopy. Similarly. we have not modeled the head domam. Obviously, mapping sites in the head domain will be very useful, since so many ribosomal proteins in these regions have been mapped. We are exploring the distribution of rRNA in these regions. In general, there is not’ a great discrepancy between other models and ours if one considers only the regions that have been localized directly by DNA-hybridizat.ion electron microscopy. One difference involves

the 518-533

loop, which


et al.

(1988a.6,c) placed on the 50 S interface side of the 30 S subunit underneath the pseudoknot structure. Our model and the Schuler & Brimacombe (1988) model place this region on the external or cytoplasmic side of the 30 S subunit. In the platform region, our model proposes a ring structure, the “platform ring”, which is closed through interactions between adjacent pairing

loops. One can easily imagine through non-Watson-Crick

similar to those between the dimethyl-U loops of tRNAs. We have




specific baseinteractions

and TUCG

to comparisons

with protein locations mapped by immunoelectron microscopy, since these have been directly related to ribosomal features such as the platform, head, cleft, neck and the ribosomal density envelope. In contrast. protein locations mapped by neutron diffraction can be related only indirectly to ribosomal “landmarks” such as the platform. That is because the neutron-mapped locations must first be compared to the immune-mapped protein locations

before they can be oriented with respect to the landmarks. This additional intermediate comparison increases the uncertainty in the neutron mappings with respect to ribosomal features. Consequently. t,he ribosomal envelope is a weak




constraint when rRNA models, such as those presented by Sternet al., (1988a,b,c), use the neutron protein data. For these reasons, we have used DNAhybridization microscopy with immuno-protein mapping data. A minor difference between the models concerns the orientation of the penultimate helix. Only one end of this helix has been localized, by DNA-hybridization microscopy, in the cleft of the subunit, and the location of the loop 1450-1493 is unknown. Most models predict that the helix extends into the base but no lateral constraints have been placed on it (as pointed out by Stern et al., 1988a,b,c). Another

helix with a relatively unconstrained orientation is the 588-651 helix. Again, it extends into the base in most models. We have based its location principally on coaxial stacking of it on helix 584-5871754-757. Finally, a minor, but unusual, structure is the antiparallel positioning of single-stranded regions, 812-816 and 570-574. We have indicated the paths of these as two single strands lying side by side. A non-Watson-Crick antiparallel interaction is possthe palindromic, phylogenetically ible, through conserved sequences, 57 1UAAA574 with 813UAAA816, but there is insufficient variability in these regions of the rRNA to test this properly. As a result of our experiments, we cannot help but note that the regions that have been most accessible to DNA probes are highly conserved and generally important for ribosome function (Moazed & Noller, 1986). We hope to gather more information concerning the rRNA tertiary structure by finding more accessible regions and by genetically engineering rRNA (Scheinman et al., 1988) for use with DNA-hybridization electron microscopy. We thank Marian Peris for photography. This work was supported by research grants from the National Science Foundation (PCM 83-16926) and the National Institute for General Medical Science (GM24034) to *J.A.L.

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et al.

by D. DeRosier

DNA-hybridization electron microscopy tertiary structure of 16 S rRNA.

Seven regions of 16 S rRNA have been located on the surface of the 30 S ribosomal subunit by DNA-hybridization electron microscopy. This information h...
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