Interaction of the Sarcin/Ricin Domain of 23 S Ribosomal RNA with Proteins L3 and L6*

Toshio UchiumiDagger §, Naoyuki SatoDagger , Akira Wada, and Akira HachimoriDagger

From the Dagger  Institute of High Polymer Research, Faculty of Textile Science and Technology, Shinshu University, Ueda 386-8567 and the  Department of Physics, Osaka Medical College, Takatsuki 569-0084, Japan

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

We investigated interaction of an RNA domain covering the target site of alpha -sarcin and ricin (sarcin/ricin domain) of Escherichia coli 23 S rRNA with ribosomal proteins. RNA fragments comprising residues 2630-2788 (Tox-1) and residues 2640-2774 (Tox-2) of 23 S rRNA were transcribed in vitro and used to analyze the binding proteins by gel shift and filter binding. Protein L6 bound to both Tox-1 (Kd: 0.31 µM) and Tox-2 (Kd: 0.18 µM), and L3 bound only to Tox-1 (Kd: 0.069 µM) in a solution containing 10 mM MgCl2 and 175 mM KCl at 0 °C. Footprinting studies were performed using the chemical probe dimethyl sulfate on full-length 23 S rRNA. Binding of L6 protected a single base, A-2757, and strongly enhanced reactivity of C-2752. A direct role of A-2757 in the L6 binding was verified by site-directed mutagenesis; replacements of A-2757 with G and C impaired the L6 binding. On the other hand, binding of L3 protected A-2632, A-2634, A-2635, A-2675, A-2726, A-2733, A-2749, and A-2750. Interestingly, binding of L6 and L3 together protected additional bases A-2657, A-2662, C-2666, and C-2667 in the sarcin/ricin loop, in addition to A-2740, A-2741, A-2748, A-2753, A-2764, A-2765, and A-2766 in the other stem-loop. This appears to be due to cooperative interaction of L3 and L6 with the RNA. The results are discussed with respect to conformational modulation of the sarcin/ricin domain by the protein binding.

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

It is generally recognized that rRNA plays a fundamental role in translation and associated ribosomal proteins help it by modulating the conformation and dynamics of rRNA (reviewed in Ref. 1). Detailed knowledge of rRNA-protein interaction is, therefore, important to understand the mechanism of action of rRNA, particularly of the conserved functional regions. The sarcin/ricin loop comprising residues 2653-2667 in domain VI of Escherichia coli 23 S rRNA is one of the most notable regions, since this includes an important site of interaction of elongation factors EF-G1 and EF-Tu with the ribosome (2-4) and also the site of action for ribotoxins, alpha -sarcin and ricin (5, 6). It is not clear whether a certain ribosomal protein interacts with this region and affects the conformation in the ribosome, although a model of the three-dimensional structure of this loop region has been built up on the basis of NMR data (7, 8).

There is evidence suggesting that assembly of ribosomal proteins to 23 S rRNA changes a feature of the sarcin/ricin loop region; (a) many sites at or near the loop region in the naked rRNA are much more reactive with a variety of chemicals and ribonucleases than those in the 50 S subunit (9), and (b) this loop region is accessible to ricin in the naked 23 S rRNA, but not in the intact E. coli ribosome (6). Leffer et al. (9) identified proteins L3 and L6 that bound to domain VI comprising residues 2629 to the 3' end of 23 S rRNA. By a footprinting approach, they localized the major binding site for L3 on the 2630-2644/2771-2788 stem, a root of the large RNA domain including the sarcin/ricin loop region, but failed to determine the L6 binding site (9). Although the binding affinity of L6 to rRNA is low (9, 10), this ability is supported by recent crystallographic data on this protein that show a domain structure homologous with a large family of RNA binding proteins (11).

Protein L6 is interesting with respect to the location and function in the ribosome. Data on immunoelectron microscopy and protein-protein cross-linking indicate that L6 is located in a position close to the base of L7/L12 stalk and to proteins L10 and L11 (12-14), and this area is mapped out as the binding sites for EF-G (15, 16) and EF-Tu (17, 18) in the ribosome. In fact, L6 cross-links to EF-G (19). These lines of evidence suggest that L6 is one of the members constructing the factor binding site in the ribosome. We here investigate the interaction of the RNA domain containing the sarcin/ricin loop region of 23 S rRNA (termed here the sarcin/ricin domain) with ribosomal proteins by gel retardation and footprinting, and demonstrate that L6 directly binds to a site within residues 2640-2774 of 23 S rRNA. By footprinting, we also show that L6, together with L3, protects 4 bases in the sarcin/ricin loop against chemical modification. Our data provide valuable information about features of the sarcin/ricin domain in the ribosome.

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Plasmid Construction and in Vitro RNA Synthesis-- The DNA fragments comprising residues 2630-2788 (Tox-1), and residues 2640-2774 (Tox-2) (see Fig. 4) were amplified using the polymerase chain reaction (20), and inserted into HindIII and XbaI sites of an expression vector, pSPT18 (Boehringer Mannheim). Base substitutions of A-2757 with G and C in Tox-2 were performed by oligonucleotide-directed mutagenesis in polymerase chain reaction (21) using primers containing the individual mutations. DNA sequences of the obtained constructs were verified by dideoxy sequencing (22). The plasmid construction for the RNA fragment containing residues 1029-1127 (corresponds to the GTPase domain) of 23 S rRNA was as described previously (23). The RNA fragments were synthesized in a solution (200 µl) containing 1,000 units of SP-6 RNA polymerase, 4 µg of template DNA linearized at the XbaI site, 40 mM Tris-HCl, pH 8.0, 14 mM MgCl2, 10 mM NaCl, 5 mM dithiothreitol, 1 mM spermidine, 10 µg of bovine serum albumin, 2 mM each of ATP, GTP, and CTP, and 0.5 mM UTP (supplemented with 50 µCi of [alpha -32P]UTP). The transcripts were purified by gel filtration on a Sephadex G-50 column (Amersham Pharmacia Biotech).

Ribosomes and Ribosomal Proteins-- The large ribosomal subunits were prepared from E. coli Q13, as described previously (24). Total proteins (TP50) extracted from the subunits (25) were fractionated by a stepwise elution from CM-cellulose (Whatman) column equilibrated with a buffer (7 M urea, 5 mM 2-mercaptoethanol, and 20 mM sodium acetate, pH 4.6) using increasing concentrations of LiCl (M), 0.04, 0.075, 0.1, 0.15, 0.2, 0.25, and 0.3. A part of each fraction was dialyzed against the renaturation buffer consisting of 0.35 M KCl, 5 mM 2-mercaptoethanol, and 20 mM Tris-HCl, pH 7.5, and tested for binding to Tox-1 by gel retardation, as described below. Protein fractions containing the binding activity were further purified by high performance ion-exchange chromatography; the protein fractions were applied to a CM-5PW column (Tosoh) equilibrated with a buffer consisting of 6 M urea, 20 mM sodium phosphate, pH 6.5, 80 mM LiCl, and 5 mM 2-mercaptoethanol, and eluted with a linear gradient of 80-280 mM LiCl. The isolated proteins were concentrated with Centricon-10 (Amicon), dialyzed against the renaturation buffer, and tested for the binding to Tox-1. Identification of the isolated proteins were performed by two-dimensional polyacrylamide gel electrophoresis (24) and amino acid sequencing of the N termini (Applied Biosystems).

Gel Retardation Assays-- A solution (5 µl) containing 5 pmol of the [32P]RNA fragments, 20 mM MgCl2, 0.35 M KCl, 30 mM Tris-HCl, pH 7.5, was preincubated at 40 °C for 20 min. After addition of 5 µl of 30 mM Tris-HCl, pH 7.5, and a protein sample as indicated in the figure legends, the mixture was incubated further at 30 °C for 10 min. RNA-protein binding was examined by electrophoresis in 6% polyacrylamide gel (acrylamide/bisacrylamide ratio of 40:1) at 6.5 V/cm, with buffer recirculation at 4 °C. The two buffer systems were used in gel analyses; system 1 contained 5 mM MgCl2, 50 mM KCl, and 50 mM Tris-HCl, pH 7.6, and system 2 contained 4 mM MgCl2, and 20 mM Tris-boric acid, pH 7.6.

Filter Binding Assays-- Radiolabeled RNA fragments (5 pmol for L6 binding and 1 pmol for L3 binding) were preincubated at 40 °C for 20 min in 50 µl of 350 mM KCl, 20 mM MgCl2, 30 mM Tris-HCl, pH 7.5. By mixing with 30 mM Tris-HCl, pH 7.5, and a protein sample, the solution was adjusted to 100 µl. The mixture was incubated for another 10 min at 30 °C, and then placed on ice for 10 min. The reaction mixture was filtered through a nitrocellulose membrane (Millipore, type HA, 0.45-µm pore size, 25-mm diameter) as described by Draper et al. (26). The filter was counted for 32P using a Beckman LS6000IC Analyzer. A background determined by filtration in the absence of proteins was subtracted from each assay. The data were fitted by nonlinear least-squares analysis with a hyperbolic binding function using Prism 2 (Graphpad Software, San Diego, CA), and the Kd values were derived from three experiments.

DMS Footprinting-- The 23 S rRNA were prepared from the isolated 50 S subunits by phenol extraction and sucrose gradient centrifugation. The 23 S rRNA (20 pmol) was preincubated at 40 °C for 10 min in 25 µl of solution containing 20 mM MgCl2, 350 mM KCl, 50 mM potassium cacodylate, pH 7.2. After adding protein samples, 50 pmol of L3, 100 pmol of L6, or both the proteins, and 50 mM potassium cacodylate, pH 7.2, the solution (50 µl) was incubated at 30 °C for 10 min. Chemical modification was started by addition of DMS (1 µl; 1:4 dilution in ethanol), followed by incubation at 30 °C for 15 min. The modified RNA samples were recovered and used as templates of primer extension, as described by Moazed and Noller (27). The used primers were 5'-GGAGAACTCATCTCGGGG-3' and 5'-GTCGTCGTCTTCAACGTT-3' complementary to residues 2771-2788 and 2813-2830 of 23 S rRNA, respectively.

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Synthetic RNA fragments that mimic the local structure of rRNA have been widely employed to investigate RNA-protein interactions (4, 9, 23, 28, 29). In the present study, we synthesized two RNA fragments covering the sarcin/ricin domain of E. coli 23 S rRNA to investigate the interaction of the RNA region with 50 S ribosomal proteins: Tox-1 comprising residues 1630-1788 and Tox-2 comprising residues 2640-2774 (see Fig. 4). Protein binding to the RNA fragments was assayed by gel retardation for each protein fraction separated with CM-cellulose column and for high performance liquid chromatography-purified proteins (see "Materials and Methods"). Two proteins showed ability to interact with Tox-1. They were identified as L3 and L6 by two-dimensional polyacrylamide gel electrophoresis and amino acid sequencing of the N termini (data not shown). Fig. 1A shows the purity of isolated L3 (lane 2) and L6 (lane 3) samples used for the binding experiments. As shown in Fig. 1B, L3 strongly bound to Tox-1 (lane 2), but not to Tox-2 (lane 5) or to a control RNA fragment containing residues 1029-1127 covering the GTPase domain (lane 8). L6 protein, on the other hand, showed a weak affinity for both Tox-1 (lane 3) and Tox-2 (lane 6), but not for the GTPase domain (lane 9). This gel condition containing 50 mM KCl (system 1, Fig. 1B) did not detect the stable L6-RNA complex, but another gel system excluding KCl (system 2, Fig. 1C) showed the L6-RNA complex as a clear band with Tox-1 (lanes 3 and 4), and Tox-2 (lanes 7 and 8) depending on concentrations of added L6. No complex was detected between L6 and the GTPase domain even in system 2 (see lanes 10-12).


View larger version (84K):
[in this window]
[in a new window]
 
Fig. 1.   Binding of the isolated L3 and L6 to the sarcin/ricin RNA domains. A, a total 50 S ribosomal protein (15 µg, lane 1) and the isolated proteins L3 (1 µg, lane 2) and L6 (1 µg, lane 3) were analyzed by 17% SDS-polyacrylamide gel electrophoresis. B, RNA fragments Tox-1 (lanes 1-3) and Tox-2 (lanes 4-6) comprising residues 2630-2788 and 2640-2774, respectively (see also Fig. 4), and the region of residues 1029-1127 corresponding to the GTPase domain (lanes 7-9) of 23 S rRNA were incubated without protein (lanes 1, 4, and 7), or with 10 pmol of isolated L3 (lanes 2, 5, and 8) and 20 pmol of L6 (lanes 3, 6, 9), as described under "Materials and Methods." The samples were then analyzed on a 6% polyacrylamide gel in buffer system 1 containing 50 mM KCl. C, Tox-1 (lanes 1-4), Tox-2 (lanes 5-8), and the GTPase domain (lanes 9-12) were incubated without protein (lanes 1, 5, and 9), or with 5 pmol (lanes 2, 6, and 10), 10 pmol (lanes 3, 7, and 11), and 20 pmol (lanes 4, 8, and 12) of L6. The samples were then analyzed on a 6% polyacrylamide gel in buffer system 2 excluding KCl (see "Materials and Methods").

Binding affinities of L3 and L6 for the RNAs were examined by filter binding assay (Fig. 2). The RNA fragments Tox-1, Tox-2, and GTPase domain were titrated with proteins L3 (Fig. 2A) and L6 (Fig. 2B). L3 bound to Tox-1 with a high affinity (Kd: 0.069 ± 0.025 µM), whereas L6 bound to Tox-1 and Tox-2 with lower affinities (Kd: 0.31 ± 0.10 µM and 0.18 ± 0.06 µM, respectively). There was no appreciable binding either of L3 to Tox-2 or the GTPase domain or of L6 to the GTPase domain. These binding data suggest that the primary binding site for L6 lies within Tox-2 and that for L3 in the 2630-2639/2775-2788 region, which remains in Tox-1 and is deleted in Tox-2.


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 2.   Titrations of L3 (A) and L6 (B) to the RNA fragments Tox-1 and Tox-2. Increasing concentrations of L3 (A) and L6 (B) were incubated with [32P-]RNA fragment: Tox-1 (bullet ), Tox-2 (open circle ), and the region of residues 1029-1127 corresponding to the GTPase domain (black-square). Each reaction mixture was applied to a nitrocellulose filter. Binding is expressed as a ratio of radioactivity retained on the filter to the input (fraction of RNA bound) in each concentration of the proteins. Data were fitted to a hyperbolic binding function (Graphpad Prism 2). The Kd values shown in the text are mean ± S.E. for three experiments.

Binding sites for L3 and L6 in 23 S rRNA were further analyzed by DMS-footprinting. Sites of the chemical modification (at N-1 of adenine and N-3 of cytosine) in the presence or absence of proteins were localized by primer-extension, followed by gel electrophoresis (Fig. 3). Binding of L3 protected bases A-2632, A-2634, A-2635, A-2675, A-2726, A-2733, A-2749, and A-2750 and enhanced the modification at A-2734 (lane 2). These bases lie in the 2630-2643/2771-2788 stem, the 2675-2733 stem, and the 2747-2757 loop (Fig. 4). Binding of L6 caused marked protection at A-2757 and enhancement at C-2752 (Fig. 3, lane 3) within the conserved loop region of residues 2747-2757 (Fig. 4). L6 also enhanced C-2699 weakly, but obviously. No effect was observed in the sarcin/ricin loop comprising residues 2653-2667 by the individual bindings. However, additions of L3 and L6 together newly caused strong protection of 4 bases in this loop region, A-2657, A-2662, C-2666, and C-2667, in addition to A-2740, A-2741, A-2748, A-2753, A-2764, A-2765, and A-2766 in the stem-loop of residues 2735-2769 (Figs. 3, lane 4, and 4). We infer that these effects given by the combination of L3 and L6 reflect a cooperative interaction of the two proteins with the RNA.


View larger version (76K):
[in this window]
[in a new window]
 
Fig. 3.   Effect of binding of L3 and L6 to 23 S rRNA on reactivity with DMS at or near the sarcin/ricin loop region. The 23 S rRNA was incubated without protein (lanes 1 and 5) and with L3 (lane 2), L6 (lane 3), and both L3 and L6 (lane 4), as described under "Materials and Methods." The samples were then incubated with DMS (lanes 1-5). The position and extent of chemical modification were analyzed by primer extension with reverse transcriptase, followed by gel electrophoresis. Primers used were for residues 2771-2788 (left panel) and for residues 2813-2830 (right panel) of individual 23 S rRNA samples. Lanes C, U, A, and G, samples terminated with respective dideoxynucleotides. Lane K, a sample of unmodified protein-free RNA. Numbers of residues of E. coli 23 S rRNA are given to the left. Arrowheads are given to the positions showing marked protein-dependent effects on DMS-modification: open arrowheads, bases protected or enhanced by L3 binding; gray arrowheads, bases protected or enhanced by L6 binding; filled arrowheads, bases markedly protected by L3 + L6.


View larger version (26K):
[in this window]
[in a new window]
 
Fig. 4.   Summary of DMS-footprinting data on the protein binding to the sarcin/ricin RNA domain. Positions where the protein binding affects reactivity with DMS are marked with circles (protection) and diamonds (enhancement) on secondary structure based on the model of Gutell et al. (39). Open symbols, affected by L3 binding; gray symbols, affected by L6 binding; filled symbols, markedly protected by L3 + L6. The regions of RNA fragments Tox-1 and Tox-2 transcribed in vitro and used in this study are indicated.

Despite the physical binding of L6 to Tox-2 (Figs. 1 and 2), our DMS-footprinting data showed protection of only A-2757 in this region (Fig. 3). To clarify whether A-2757 is involved in L6 binding, we performed a site-directed mutagenesis. Effect of base substitutions of A-2757 with G and C on L6 binding to Tox-2 was tested by gel retardation in system 2. As shown in Fig. 5, the binding was disrupted by either A to C transversion (lane 4) or A to G transition (lane 6). These results combined with the footprinting data suggest that A-2757 plays a direct role in L6 binding to the RNA.


View larger version (50K):
[in this window]
[in a new window]
 
Fig. 5.   Effect of mutations at position 2757 on L6 binding. The transcripts of wild-type Tox-2 (lanes 1 and 2), the A-2757right-arrowC variant (lanes 3 and 4), and the A-2757right-arrowG variant (lanes 5 and 6) were incubated without (lanes 1, 3, and 5) or with 20 pmol of L6 (lanes 2, 4, and 6). The samples were then analyzed by gel retardation in system 2, as shown in Fig. 1C.


    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Interaction of the Sarcin/Ricin RNA Domain with Ribosomal Proteins-- Little has been known about ribosomal proteins assembled to an RNA domain including the conserved sarcin/ricin loop region. The present study demonstrates that protein L6 directly binds to a limited region within residues 2640-2774 (Tox-2). The marked effects of L6 binding on DMS modification is localized in the conserved loop of residues 2747-2757: protection of A-2757 and enhancement of C-2752 (Fig. 4). In addition, replacement of this protected base A-2757 with G or C causes disruption of L6 binding (Fig. 5). The results suggest that L6 recognizes a local structure including A-2757 within Tox-2 as a primary binding site. The present results are in line with previous data on RNA binding ability of L6 to the 3' half (10) and to domain VI of 23 S rRNA (9), and also consistent with cross-linking between a mammalian homologue (L9) of E. coli L6 and ricin A chain, which attacks the sarcin/ricin loop (30). On the other hand, L6 is cross-linked to a region of residues 2473-2481 of domain V of 23 S rRNA with 2-iminothiolane (31), although direct binding of L6 to an RNA fragment for domain V was not detected (9). It is presumed that the 2473-2481 region of domain V lies in the close proximity to, if not directly interacts with, L6 in the ribosome.

Another protein, L3, strongly binds to Tox-1, but not to Tox-2, suggesting that the region of residues 2630-2639/2775-2788 lacking in Tox-2 is the major binding site for L3. This is consistent with previous footprinting data by Leffers et al. (9). In our footprinting study, L3 protects not only 3 bases in the 2630-2639/2775-2788 region, but also 5 bases in Tox-2 region (Fig. 4). Therefore, together with the 2630-2639/2775-2788 region, some parts within Tox-2 may constitute L3 binding site.

Interestingly, our footprinting data also reveal additional protections at the sarcin/ricin loop by the combination of L3 and L6; bases A-2657, A-2662, C-2666, and C-2667 in the sarcin/ricin loop in addition to 7 bases in another stem-loop of residues 2735-2769 are protected by binding of L3 and L6 together (Fig. 4). All of these positions are not reactive with DMS within the 50 S subunit (data not shown), suggesting that the present data reflect the structural feature of the sarcin/ricin domain in the intact ribosome. It is not clear, however, at present whether these additional protections are caused by contact with protein(s) or conformational change of the RNA induced by the two proteins. L3 is known as one of the important proteins bound earliest during the subunit assembly (32) and expected to induce a conformational change of 23 S rRNA, which leads to the next step of the 50 S subunit assembly. Therefore, it is most likely that L3 binding affects a conformation of the Tox-2 region, although there is no detectable effect of L3 alone on the DMS modification at the sarcin/ricin loop. From these considerations, we deduce a tentative model of the RNA folding: the 2630-2644/2771-2788 stem and the 2675-2732 stem fold close together in one side by the strong L3 binding, and in the other side, the sarcin/ricin loop region and the 2735-2769 stem-loop region become closer as much as L6 interacts with both regions and protects them. Further extensive investigations are required to elucidate the mechanism of this cooperative interaction of L3 and L6 with the RNA.

Position of the Sarcin/Ricin RNA Domain in the Ribosome-- The position of L6 near the base of the L7/L12 stalk in the 50 S ribosomal subunit has been established from immunoelectron microscopy (12, 13) and protein-protein cross-linking studies (14). In addition, this area underneath the L7/L12 stalk has been observed as the binding site for EF-G (15, 16) and EF-Tu (17, 18). The present results demonstrate that the sarcin/ricin RNA domain (Tox-2) of residues 2640-2774 in 23 S rRNA interacts directly with L6, implying that this RNA domain is situated in the vicinity of L6 at the factor binding site for EF-G and EF-Tu in the ribosome. This is consistent with the facts that EF-G is cross-linked to L6 (19) and that both of EF-G and EF-Tu protects the sarcin/ricin loop from chemical modification (3).

Besides the sarcin/ricin domain, another conserved RNA domain termed the GTPase domain (or thiostrepton binding site) in domain II of 23 S rRNA (33-36) is known to interact with EF-G (3, 4). These two RNA domains appear to collaborate in EF-G-dependent process of translation, although the two domains are distant in the primary and secondary structure of 23 S rRNA. It is of interest to see the topographical relationship between the sarcin/ricin domain and the GTPase domain on a model of protein topography (Fig. 6). Since the sarcin/ricin domain is situated near L6 (present study) and the GTPase domain on L11 (28, 33), the two RNA domains are expected to be adjacent to each other. This view is strongly supported by recent evidence from directed hydroxyl radical probing for EF-G binding site (37), i.e. both the sarcin/ricin domain and the GTPase domain are cleaved by the same EF-G conjugated with Fe (II) at position 650 of the molecule. The vicinity of these two RNA domains may explain the fact that binding of thiostrepton to the GTPase domain inhibits accessibility of the sarcin/ricin loop for alpha -sarcin (38).


View larger version (29K):
[in this window]
[in a new window]
 
Fig. 6.   Possible position of the sarcin/ricin domain neighboring to the GTPase domain (the thiostrepton binding site) in the ribosome. The RNA regions of residues 2640-2774 (Tox-2) and residues 1051-1108 are placed on L6 and L11, respectively, of a model of the protein topography for the 50 S subunit, presented by Walleczek et al. (13).


    ACKNOWLEDGEMENTS

We thank Dr. R. Kominami (Niigata University) for helpful discussion and critical reading of the manuscript and Dr. S. Odani (Niigata University) for helpful advice on the binding experiments.

    FOOTNOTES

* This work was supported by Grant-in-aid for Scientific Research 10174212 and Grant-in-aid for COE Research 10CE2003 from the Ministry of Education, Science, Sports and Culture of Japan, and by Grant JSPS-RFTF96100305 from the Japan Society for the Promotion of ScienceThe costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed. Fax: 81-268-21-5571; E-mail: uchiumi{at}giptc.shinshu-u.ac.jp.

The abbreviations used are: EF, elongation factor; DMS, dimethyl sulfate.
    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

  1. Noller, H. F. (1991) Annu. Rev. Biochem. 60, 191-227[CrossRef][Medline] [Order article via Infotrieve]
  2. Hausner, T. P., Atmadja, J., and Nierhause, K. H. (1987) Biochimie (Paris) 69, 911-923[CrossRef][Medline] [Order article via Infotrieve]
  3. Moazed, D., Robertson, J. M., and Noller, H. F. (1988) Nature 334, 362-364[CrossRef][Medline] [Order article via Infotrieve]
  4. Munishkin, A., and Wool, I. G. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 12280-12284[Abstract/Free Full Text]
  5. Endo, Y., and Wool, I. G. (1982) J. Biol. Chem. 257, 9054-9060[Abstract/Free Full Text]
  6. Endo, Y., and Tsurugi, K. (1988) J. Biol. Chem. 263, 8735-8739[Abstract/Free Full Text]
  7. Szewczak, A. A., Moore, P. B., Chan, Y.-L., and Wool, I. G. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 9581-9585[Abstract]
  8. Szewczak, A. A., and Moore, P. B. (1995) J. Mol. Biol. 247, 81-98[CrossRef][Medline] [Order article via Infotrieve]
  9. Leffers, H., Egebjerg, J., Andersen, A., Christensen, T., and Garrett, R. A. (1988) J. Mol. Biol. 204, 507-522[CrossRef][Medline] [Order article via Infotrieve]
  10. Spierer, P., Zimmermann, R. A., and Mackie, G. A. (1975) Eur. J. Biochem. 52, 459-468[Abstract]
  11. Golden, B. L., Ramakrishnan, V., and White, S. W. (1993) EMBO J. 12, 4901-4908[Abstract]
  12. Stöffler-Meilicke, M., Noah, M., and Stöffler, G. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 6780-6784[Abstract]
  13. Walleczek, J., Schüler, D., Stöffler-Meilicke, M., Brimacombe, R., and Stöffler, G. (1988) EMBO J. 7, 3571-3576[Abstract]
  14. Traut, R. R., Tewari, D. S., Sommer, A., Gavino, G. R., Olson, H. M., and Glitz, D. G. (1985) in Structure, Function and Genetics of Ribosomes (Hardesty, B., and Kramer, G., eds), pp. 286-308, Springer-Verlag, New York
  15. Girshovich, A. S., Kurtskhalia, T. V., Ovchinnikov, Y. A., and Vasiliev, V. D. (1981) FEBS Lett. 130, 54-59[CrossRef][Medline] [Order article via Infotrieve]
  16. Agrawal, R. K., Penczek, P., Grassucci, R. A., and Frank, J. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 6134-6138[Abstract/Free Full Text]
  17. Girshovich, A. S., Bochkareva, E. S., and Vasiliev, V. D. (1986) FEBS Lett. 197, 192-198[CrossRef][Medline] [Order article via Infotrieve]
  18. Stark, H., Rodnina, M. V., Rinke-Appel, J., Brimacombe, R., Wintermeyer, W., and van Heel, M. (1997) Nature 389, 403-406[CrossRef][Medline] [Order article via Infotrieve]
  19. Sköld, S.-E. (1982) Eur. J. Biochem. 127, 225-229[Abstract]
  20. Saiki, R. K., Gelfand, D. H., Stoffel, S., Share, S. J., Higuchi, R., Hoen, G. T., Mullis, K. B., and Erlich, H. A. (1988) Science 239, 487-491[Medline] [Order article via Infotrieve]
  21. Higuchi, R., Krummel, B., and Saiki, R. K. (1988) Nucleic Acids Res. 16, 7351-7367[Abstract]
  22. Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 5463-5467[Abstract]
  23. Uchiumi, T., Wada, A., and Kominami, R. (1995) J. Biol. Chem. 270, 29889-29893[Abstract/Free Full Text]
  24. Wada, A. (1986) J. Biochem. (Tokyo) 100, 1583-1594[Abstract]
  25. Hardy, S. J. S., Kurland, C. G., Voynow, P., and Mora, G. (1969) Biochemistry 8, 2897-2904[Medline] [Order article via Infotrieve]
  26. Draper, D. E., Deckman, I. C., and Vartikar, J. V. (1988) Methods Enzymol. 164, 203-220[Medline] [Order article via Infotrieve]
  27. Moazed, D., and Noller, H. F. (1986) Cell 47, 985-994[Medline] [Order article via Infotrieve]
  28. Ryan, P. C., Lu, M., and Draper, D. E. (1991) J. Mol. Biol. 221, 1257-1268[CrossRef][Medline] [Order article via Infotrieve]
  29. Uchiumi, T., Traut, R. R., Elkon, K., and Kominami, R. (1991) J. Biol. Chem. 266, 2054-2062[Abstract/Free Full Text]
  30. Vater, C. A., Bartle, L. M., Leszyk, J. D., Lambert, J. M., and Goldmacher, V. S. (1995) J. Biol. Chem. 270, 12933-12940[Abstract/Free Full Text]
  31. Wower, I., Wower, J., Meinke, M., and Brimacombe, R. (1981) Nucleic Acids Res. 9, 4285-4302[Abstract]
  32. Nowotny, V., and Nierhaus, K. H. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 7238-7242[Abstract]
  33. Schmidt, F. J., Thompson, J., Lee, K., Dijk, J., and Cundliffe, E. (1981) J. Biol. Chem. 256, 12301-12305[Abstract/Free Full Text]
  34. Beauclerk, A. A. D., Cundliffe, E., and Dijk, J. (1984) J. Biol. Chem. 259, 6559-6563[Abstract/Free Full Text]
  35. Egebjerg, J., Douthwaite, S., Liljas, A., and Garrett, R. A. (1990) J. Mol. Biol. 213, 275-288[Medline] [Order article via Infotrieve]
  36. Rosendahl, G., and Douthwaite, S. (1993) J. Mol. Biol. 234, 1013-1020[CrossRef][Medline] [Order article via Infotrieve]
  37. Wilson, K. S., and Noller, H. (1998) Cell 92, 131-139[Medline] [Order article via Infotrieve]
  38. Miller, S. P., and Bodley, J. W. (1991) Nucleic Acids Res. 19, 1657-1660[Abstract]
  39. Gutell, R. R., Larsen, N., and Woese, C. R. (1994) Microbiol. Rev. 58, 10-26[Abstract]


Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.