Biochimie (1991) 73,961-969
© Soci6t6 franqaise de biochimie et biologie mol6culaire / Elsevier, Pads
961
A consonant model of the tRNA-ribosome complex during the elongation cycle of translation J Wower, RA Zimmermann* Department of Biochemistry, University of Massachusetts, Amherst, Massachusetts 01003, USA
(Received 26 January 1991; accepted 13 March 1991)
Summary - - Chemical and photochemical affinity techniques have been used extensively to determine the positions of the tRNA binding sites on the Escherichia coil ribosome. Recent advances in our understanding of ribosome structure and function prompted us to critically review the data that have accumulated on tRNA-ribosome cross-links. As a result, we propose a new model of the tRNAribosome complex that accounts for nearly all of the pertinent evidence. affinity labeling / cross-linking techniques / tRNA-ribosome complex / peptidyi transferase center / decoding site
Introduction
tRNA binding sites on the ribosome
During the last 20 years, numerous cross-linking studies designed to locate the tRNA binding sites on the Escherichia coli ribosome have been carried out [1-5]. Over the same period, our understanding of the structure and function of the ribosome has advanced significantly. Almost all of the ribosomal proteins have now been mapped on the surface of the ribosomal subunits by immune electron microscopy [6, 7]. In addition, the 3-dimensional organization of the proteins in the 30S subunit has been defined by neutron scattering [8]. While similar studies on the 50S subunit are not yet complete, important insights into the spatial relationships among 50S-subunit proteins have been gained by combining proteinprotein cross-linking results with those derived from immune electron microscopy [6, 9]. Furthermore, the development of techniques for studying the intra- and intermolecular interactions of large RNAs has le6 to the construction of rather detailed 3-dimensional models of the 16S rRNA [10, 11] and a portion of the 23S rRNA [12], together with their associated proteins. These advances in our knowledge of ribosome structure prompted us to re-examine the available data on tRNA-ribosome cross-linking. The result is a new model of the tRNA-ribosome complex that is consistent with most of the pertinent structural information.
In the original model of the translating ribosome prop,~sed by Watson [13], ribosomes were envisioned to have only two tRNA binding sites, a donor (D) site for the tRNA donating its peptidyl moiety to the growing polypeptide chain and an acceptor (A) site for the aminoacyl-tRNA accepting this moiety during formation of the peptide bond. The donor (D) and acceptor (A) sites are now generally known as the peptidyl (P) and aminoacyl (A) sites, respectively (fig l a). These two sites are functionally distinct, as only peptidyl-tPNA bound to the P site is able to react with puromycin, an antibiotic which acts as an analog of the terminal aminoacyl-adenosine portion of A sitebound aminoacyl-tRNA [14]. This two-site model was later modified to include two more tRNA binding sites (fig lb). One of them, initially proposed by Wettstein and Noll [15] and now called the exit (E) site, receives deacylated tRNA from the P site prior to its release from the ribosome. Convincing evidence for the existence of this site has been provided by several groups [ 16-19] and its role in maintaining translational accuracy has recently been substantiated [20]. The presence of another site, the recognition (R) site, to which the aminoacyl-tRNA.EF-Tu.GTP ternary complex binds, was originally proposed by Hardesty et al [21] and later incorporated into the models of Lake [22] and Johnson et al [23]. Recent studies by Noller et al have raised the possibility that there are two intermediate states, designated P/E and A/P to emphasize their hybrid character
*Correspondence and reprints
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J Wower, RA Zimmermann
(a)
(b)
(c)
Fig 1. Evolution of the concept of tRNA binding sites on the ribosome, a) The two-site model of Watson [13]; b) the tbur-site model; c) the four-site model with intermediate states added. A, aminoacyl site; P, peptidyi site; R, recognition site; E, exit site. The A/P and P/E sites of Moazed and Noller [24] correspond to At, and Ph sites of Abdurashidova et al [251, respectively (see text).
124], which are occupied by tRNAs immediately after peptide bond formation (fig l c). In the P/E site, the anticodon of the tRNA is stabilized in the P site through base pairing with the mRNA codon, while the 3' end is located in the 50S-subunit E site. In a similar vein, the anticodon of tRNA in the A/P site is thought to remain in the A site, while the 3' end, carrying the peptidyl moiety, has been transferred to the P site. According to this view, the R, A, P and E sites are designated the Aft, A/A, P/P and E sites, respectively. The six-state model of Moazed and Noller [241 closely resembles a model proposed three years earlier by Budowsky et al [25]. Here, the R, A, P and E sites are called Ar, A~, Pt (or Pr) and S, respectively. In addition to these four sites, two additional sites, Ab and Pb, correspond to the A/P and P/E sites. Given that the data concerning sites other than R, A, P and E come only from Noller's and Budowsky's laboratories, the designations proposed by these authors will be used in what follows to avoid confusion. Strategies f o r cross-linking tRNA to the ribosome
Cross-linking provides a straightforward means for !dentifying ribosomal components that are close to, or m contact with tRNA bound to functional ribosomal complexes. Two general approaches have been developed in the last two decades. One approach, designed for cross-linking unmodified tRNAs, relies on the direct photolysis of tRNA-ribosome complexes by UV light [24]. Irradiation at 254 nm, however, generates random cross-links between tRNA and the ribosome, as well as spurious intraribosomal cross-links that inactivate the ribosomal particles. To avoid this,
some workers have induced photolysis by irradiation with UV light at wavelengths between 300--400 nm. In general, such an approach is useful only for tRNAs containing naturally photoreactive nucleosides since the four commonly occurring nucleosides exhibit relatively low photoreactivity at these wavelengths. Because of the scarcity of tRNAs with suitably reactive residues, most cross-linking studies have utilized a second approach in which tRNAs derivatized with chemically or photochemically reactive probes [3]. In most instances, the probes have been attached either to the a-amino group of aminoacyltRNA or to one of a number of positions in tRNA occupied by naturally occurring, modified bases. In one case, the guanosine residues of tRNA have been derivatized randomly [27]. The main drawback of this approach is that the long and bulky substituents which are frequently used can potentially interfere with the proper binding of tRNA to the ribosome. In addition, the cross-links that result from the use of such probes are necessarily at some distance from the actual tRNA binding site. In an effort to avoid the shortcomings of the two approaches described above, three new strategies have been developed. In one, flavin mononucleotide (FMN) irradiated at 350 nm was used as a photosensitizer; by generating singlet oxygen, superoxide and hydroxyl radicals, the activated FMN initiates free-radical processes leading to tRNA-ribosome cross-linking [28]. However, because photosensitization in itself leads to rapid inactivation of ribosomes [29], this method is not particularly advantageous for crosslinking studies. In the second strategy, cross-linking is performed with tRNA molecules in which cytidine residues have been chemically converted to 4-thiouridine (s4U) [30, 31 ]. As this nucleoside is photoreactive at 335 nm, the thiolated tRNAs can be directly cross-linked to ribosomes without affecting their structure or function. While this approach has great potential for analyzing ribosomal components in the neighborhood of cytidine residues in ribosome-bound tRNA, suitable means must be developed to separate aminoacyltRNA from the background of non-aminoacylated tRNA and to determine the identity of the cross-linked residue(s) after photolysis. Over the past few years, we have developed a third strategy which entails the introduction of photoreactive azidonucleosides at defined positions in the tRNA molecule [32-34]. These nucleoside analogs, such as 2-azidoadenosine and 8-azidoadenosine, can in principle be inserted at any position as long as the substitution does not interfere with the function of the tRNA molecule. Cross-linking of the azidonucleosidecontaining tRNAs to ribosomes is achieved by irradiation of noncovalent tRNA-mRNA-ribosome complexes with light of 300 nm, a wavelength which
A model of the tRNA-ribosome complex neither generates spurious cross-links nor disrupts the structure or function of the ribosome. Since the photochemical reaction leads to the formation of very short cross-links (2-4 A), only those ribosomal components in close proximity to the photoreactive nucleoside become covalently attached.
Identification of ribosomal components at the peptidyi transferase center and in the decoding domain The majority of cross-linking experiments have been carried out with aminoacyl-tRNAs derivatized on their aminoacyi moiety in order to identify ribosomal component(s) at the peptidyl transferase center of the ribosome. These probes react exclusively with 50S ribosomal subunits. Among the ribosomal proteins most consistently labeled are L2, LI5, LI6 and L27 [5]. Significantly, the close proximity of these proteins within the 50S subunit has been demonstrated by inter-protein cross-linking [14, 35]. Moreover, it has been shown by immune electron microscopy [6, 36] that proteins L2, L15 and L27 are clustered on the interface of the 50S subunit in the valley between the L1 ridge and the central protuberance (fig 2a). Although the 23S rRNA was labeled by various derivatized aminoacyl-tRNAs, sites of cross-linking have only been precisely identified for 3-(4'-benzoylphenyl)propionyl-Phe-tRNAPhe [37]. In the latter case, nucleotides A2451 and C2452 were cross-linked by P site-bound tRNA while nucleotides U2584 and U2585 were crossqinked to the tRNA when it was located in the A site. A rain, :"reaction with the AUGU sequence spanning positions 2503-2506 was observed when 3(4'-bcnzoylph,:,yl)propionyl-Phe-tRNA was in either
3'
(o)
(b)
Fig 2. Models of the 30S and 50S ribosomal subunits of E coli. a) 50S subunit; the cluster of proteins most frequently labeled by aminoacyl-tRNAs derivatized on the aminoacyl moiety is cross-hatched; numbers represent positions of 50S subunit patterns as in [6, 36]. b) 30S subunit; orientation of the mRNA according to Olson et al [43]. 1400, the location of nucleotide C1400 of the 16S rRNA [40].
963
site. Interestingly, although the labeled nucleotides are not adjacent to each other in the primary structure of the 23S rRNA, they all reside in the same portion of the secondary structure which is referred to as the central loop of domain V and is often identified with the peptidyl transferase center. This structure is expected to be in close proximity to protein L2 in the 50S ribosomal subunit since nucleotide U1782, within the binding site for protein L2, has been cross-linked to nucleotide U2609, within the central loop [38]. In addition, short range cross-links have been established to proteins L2, L15, LI6 and L27, as well as to nucleotide G1945 of the 23S rRNA from yeast tRNA phe variants containing azidoadenosine at positions 76 and 73, one or four bases removed from the aminoacyl group, respectively [33]. While the cross-linking studies carried out with aminoacyl-tRNAs derivatized on their aminoacyl moiety or with azidonucleosides incorporated at or near their 3' terminus have not identified a unique ribosomal component or structure that might correspond to peptidyl transferase, they do indicate that the aminoacyl arms of the L-shaped tRNAs at both the A and P sites interact with a limited area of the 50S subunit defined by proteins L2, LI5, LI6 and L27, and portions of domains IV and V of the 23S rRNA (fig 2a). Since mRNA binds exclusively to the 30S ribosomal subunit, the decoding domain - the site or sites at which the tRNA anticodons interact with the mRNA codons - must be located on this subunit. To define this domain, affinity-labeling experiments have been performed with reactive derivatives of both tRNA and mRNA [2, 3]. In one case, photochemical cross-linking of E coil tRNA val to the 30S-subunit P site was found to be mediated by the 5' anticodon base of the tRNA and residue C1400 of the 16S rRNA [39]. Investigation of this complex by immune electron microscopy [40] demonstrated that the site of codon-anticodon interaction lies in the cleft between the head and the platform of the 30S subunit (fig 2b). Cross-links from azidoadenosines at positions 37 in the anticodon loop to protein $7, and at position 43 in the anticodon stem to proteins S13 and S19 are consistent with this localization ([41]; LA Sylvers, J Wower and RA Zimmermann, unpublished data). When mRNA analogs with affinity probes attached to their 5' or 3' termini were used to elucidate mRNAribosome interactions, several proteins, covering a large fraction of the 30S subunit surface, were labeled [3]. Consequently, these results did not permit a clear definition of the mRNA-binding region. More recent studies, employing mRNA analogs with reactive nucleosides at internal positions, have shown that proteins $7, $21 and a segment of the 16S rRNA very close to its 3' terminus are within the decoding domain [42].
964
J Wower, RA Zimmermann
A matter of critical importance for understanding the way in which tRNA molecules are positioned on the 30S subunit is the orientation of the mRNA. Two independent lines of evidence demonstrate that the mRNA crosses the cleft in a 3' to 5' direction, when viewed from the 30S subunit interface (fig 2b). In the first, mRNA analogs with antigenic substituents at either the 5' or 3' end were bound to the 30S particle, allowed to interact with the appropriate antibodies, and analyzed by immune electron microscopy [43]. In the second, the path of the mRNA was inferred from cross-links to 16S rRNA and 30S subunit proteins from synthetic mRNA analogs containing, at three different positions, a single thiouridine residue derivatized with 4-azidophenacylbromide [42]. P site-bound tRNA must be positioned so that its anticodon is anchored by the mRNA in the cleft of the 30S subunit while its aminoacyl stem is directed toward the 50S subunit. It follows that the tRNA in the A site should be similarly positioned as both the 3' termini and the anticodons of the two tRNAs must be closely juxtaposed. Because mRNA is translated in a 5'-->3' direction, the A-site tRNA must be to the left, and the Psite tRNA to the right, of the interface surface of the 30S subunit (fig 4). While it is now generally agreed that the peptidyl transferase center is located between the central protuberance and the L1 ridge of the 50S subunit and that the decoding domain is situated between the head and the platform of the 30S subunit, little is known about other contacts between tRNA and the ribosome. Models of tRNA.mRNA.ribosome complex To aid in interpreting the data on the location of the ribosomal binding sites for tRNA, several models of the tRNA-ribosome complex have been proposed in the course of the last 20 years. These will be briefly discussed below, in historical order.
INrERFACE
CAVITY
(a)
(b)
Fig 3. Model for the arrangement of tRNAs on the 50S subunit of the E coli r~bosome, a) Peptidyl (P) and aminoacyi (A) sites. (b) Exit (E), peptidyl (P) and recognition (R) sites. Numbers indicate locations of 50S subunit proteins according to [6, 36].
|
3'
Fig 4. Model for the arrangement of tRNAs in the aminoacyl (A) and peptidyl (P) sites of the 30S subunit of the E coil ribosome. Numbers indicate locations of 30S subunit proteins according to [6]. One of the first models, advanced by Lake [22], proposed locations for three tRNA molecules on the 30S subunit, at the R, A and P sites. This model was later extended to include the 50S subunit [44]. The sites of interaction of the tRNAs on the ribosome were deduced from immune electron-microscopic mapping of the ribosomal proteins and from information available at the time about the function of these proteins. In Lake's model, the R site-bound tRNA is located on the solvent side of the 30S subunit in such a way that its anticodon is anchored at the solvent end of the cleft by anticodon-codon interaction with mRNA and its Lshaped body is adjacent to proteins $5, $8, S10, S12 and S14. The anticodon of the A site-bound tRNA must be at the same location as that of the R sitebound tRNA. Based on the location of proteins crosslinked to initiation factors IF2 [45] and IF3 [46], which guide tRNA~ et to the 30S subunit, P site-bound tRNA was placed within the cleft. The relative positions of the A and P sites in this model, however, would require a 5'-+3' orientation of the mRNA across the small-subunit interface. Identification of protein S14 as a major target for puromycin cross-linking prompted Olson et al [47] to propose a slightly different model for tRNA at the A site of the 70S ribosome. While the position of the Asite tRNA in this version is very similar to that in Lake's model, the aminoacyl acceptor end of the tRNA interacts with the head rather than the platform of the 30S particle on the subunit interface. Another model for the arrangement of tRNA molecules on the ribosome was suggested by Spirin [48]. In this scheme, the anticodons of the tRNAs bound to the A and P sites are located on the solvent side of the 30S subunit, in the groove separating its head and body, and their aminoacyl acceptor ends lie in a valley, just below the central protuberance, on the
A model of the tRNA-ribosomecomplex interface side of the 50S subunit. The A site is formed in part by the portion of the 30S subunit head that encompasses proteins S14 and S 19, and in part by the central protuberance of the 50S subunit. The P site is located on the surface of the 30S subunit in the neighborhood of protein $5 and the surface of the 50S subunit at the base of the L7/L 12 stalk. Cross-linking of yeast tRNAphe with probes attached to the central portion of the L-shaped molecule led to a fourth model of the ribosomal A and P sites [5, 49]. In this model, the anticodons of both tRNAs are in the cleft while their aminoacyl ends point toward the head of the 30S subunit. The tRNAs are oriented on the 30S subunit in such a way that, after binding to the 50S subunit, the P-site tRNA is positioned in the valley between the LI ridge and the central protuberance, while the A-site tRNA is closer to the L I ridge. Here again, the locationa of the Aand P-site tRNAs do not accord with me mRNA orientation derived from immune electron microscopy and cross-linking studies [42, 43]. In the model of Gold [50], the mRNA and tRNAs are placed on opposite sides of the 30S ribosomal subunit" the mRNA binds to an open 'trough' carved into the solvent side, and the tRNAs are located in the space between the ribosomal subunits. In order to interact with mRNA, the tRNA anticodons protrude into the trough through 'slots' in the 30S subunit. The 3' ends of A- and P-site tRNAs project toward the 50S subunit to contact the peptidyl transferase center while the E-site tRNA is placed in the vicinity of the L1 ridge. The relative positions of the A, P and E sites on the ribosome are in agreement with those implied by the 3'--->5'orientation of the mRNA on the 30S subunit interface. A model recently proposed by this laboratory was based largely on photoaffinity labeling studies with azidonucleoside-substituted derivatives of yeast tRNAphe [33]. Ip this depiction, the aminoacyl arms of both A- and P-site tRNAs are directed toward the peptidyl transferase center on the interface surface of the 50S subunit, while the anticodons interact with mRNA in the cleft of the 30S subunit. The A site is located to the left of the P site when viewed from the 30S subunit interface, consistent with the preferred orientation of the mRNA. A model quite similar in concept to the pre.ceding was derived from a very different expenmental approach - an analysis of alterations in the chemical reactivity of rRNA bases elicited by tRNA bound to the A, P and E sites. First advanced on the basis of results for the 30S subunit [ 11], this model was later extended to the 50S subunit by the inclusion of chemical modification data from the 23S rRNA [52]. Because the 3' terminus of E site-bound tRNA was found to protect several bases within the binding site for protein L l, these authors inferred that the E site is located between the L1 ridge and the central protuberance.
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Using 3-dimensional image reconstruction in conjunction with electron microscopy, Wagenknecht et al [53] have recently identified a large cavity, the 'interface canyon', just below the central protuberance on the interface of the 50S subunit. These authors suggest that the interface canyon could accommodate the aminoacyl stems of the A and P site-bound tRNAs. According to this model, the anticodons of the tRNAs are positioned on the surface of the 30S subunit as suggested earlier by Spirin [48]. A new model of the tRNA-ribosome complex
In this section, we develop a model for the arrangement of tRNA binding sites on the ribosome that is based mainly on tRNA-ribosome cross-linking results. The data on tRNA-ribosome cross-linking can be divided into two main categories. The first category derives from experiments that provide information not only about the labeled ribosomal components but also about the site in the tRNA molecule from which the cross-link occurred (table I). The second category only specifies the ribosomal components that were labeled [25, 27, 31]. Further consideration of the arrangement of tRNAs on the ribosome will only be based on data from the first category, as the latter are incomplete from a topographical point of view. Moreover, we have included, for the purposes of this discussion, only those experiments in which the binding site occupied by the cross-linked tRNA was satisfactorily defined. As indicated earlier, the available data favor models that: a) place the anticodons of A and P site-bound tRNAs in the cleft of the 30S ribosomal subunit; b) orient the aminoacyl arms of these tRNAs toward the L2-L15-L16-L27 protein cluster on the interface surface of the 50S subunit; and c) predict a relative arrangement of the A- and P-site tRNAs that is consistent with the 3'--->5' directionality of the mRNA across the interface surface of the 30S subunit. Although the models proposed by Wower et al [33] and Noller et al [52] fulfil these three criteria, they do not always account for the most recent cross-linking results listed in table I. For example, the cross-linking of nucleotides located both at the 3' terminus (positions 73 and 76) and within the central region (positions 17, 18, 56 and 59) of the P site-bound tRNA to proteins L2 and L27 via short-range bonds [32, 33, 54], cannot be accounted for by any model of the tRNA-ribosome complex suggested so far. To explain these data, one has to accept the notion that the aminoacyl acceptor arm of the P-site tRNA, and perforce the A-site tRNA, are immersed in a cavity on the interface side of the 50S subunit that is bounded, at least in part, by proteins L2 and L27 (fig 3a). As indicated earlier, the presence of such a cavity in the 50S subunit has already been inferred from electron microscopy [53, 55].
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Table I. tRNA-ribosome cross-links Ribosomal site
tRNA
Positwn in tRNA
Probe
Labeled component
Ref
p
tRNAphe
A76
2-4 A (azide)
L27, LI5, LI6 G 1945/23S RNA L27
[33]
2--4/~, (azide)
L27 L27, L2
[32]
14-20 ]k (300 nm)
S 19, L5, L27
[5]
G43
2-4 A (azide)
SI3, S19
[411a
t6A37
17 A 24 A
$7, LI, L27, L33 $7
[581
Y37
2-4 A (azide)
$7
cmoSU34
3-4 A (300 nm)
C 1400/16S RNA
[391
U60 u45 A21 U60 C56 Cl7 U60 C56 G44 C17
2-4 A (254 nm)
S9 $7 $5 $9 L27 L2 S9 L27 L5 L2
[59]
U59 A26 G 18 A9
2-4 A, (254 nm)
L2 $7 L27 Sl0
[54]
E coli
tRNAPhe
s4U8
9/~
S 19
[60]
A76
2-4 A, (azide)
L33
yeast
A73
p
tRNAPhe yeast
A76 A73
P
tRNAphe
acp3U47
E coli
p P P P
tRNAPhea yeast tRNA.Met yeast tRNAphe yeast tRNAIval E coil
P
tRNAphe E coli
Pt
tRNAPhe E coli
Pb
tRNAPhe E coli
Ab
A
tRNAphe
2-4/~ (254 nm) 2-4 A (254 nm)
b
[541 [54]
E coli
E
tRNAphe E coli
aAnticodon arm (residues 28-43); bLA Sylvers, J Wower, RA Zimmermann, unpublished results; cJ Wower, P Scheffer, W Wintermeyer., RA Zimmermann, unpublished result. Placement of the acceptor arms of the A- and P-site tRNAs within the interface cavity of the 50S subunit also provides a simple explanation for the persistent labeling of proteins L2 and L27 by or-amino derivatives of aminoacyl-tRNAs [5]. Moreover, this proposition suggests a way in which the ribosome could facilitate formation of the peptide bond. As no single ribosomal component exhibits high affinity toward the 3' terminus of the tRNA molecule, it is possible that the interface cavity, a feature whose structure is attributable to a number of components, guides the 3' ends of tRNAs to the peptidyl transferase center.
In our model, the 50S-subunit portion of the A site has been positioned to the right of the P site when viewed from the interface side (fig 3a). Placement of the A-site tRNA near protein LI 1 is consistent with the location of the EF-Tu binding site at the base of the L7/LI2 stalk [56]. It is also in accordance with the finding that E coli tRNAPhe, cross-linked via position 47 to EF-Tu, can bind to the A site when the P site is occupied by aminoacylated tRNA [57]. The disposition of the P site on the 30S subunit is now delineated by a number of cross-links (fig 4). As cmosU34 of E coli tRNAyal can form a cyclobutane
A model of the tRNA-ribosomecomplex dimer with C1400 of the 16S rRNA, the tRNA anticodon must lie within 3--4 A of the rRNA [39]. As mentioned above, the site of cross-linking has been mapped to the cleft of the 30S subunit [40]. The immediate surroundings of the anticodon loop have been further defined by a short-range cross-link from 2-azidoadenosine at position 37 of yeast tRNAphe to protein $7 (LA Sylvers, J Wower and RA Zimmermann, unpublished data). $7 is also the predominant protein labeled by yeast tRNA Met derivatized at position 37 with 17- and 24-A, probes [58]. Interestingly, the 17-A probe also reacts with protein L I in the 50S subunit. This result is consistent with the proximity of $7 and LI in the 70S ribosome inferred from proteinprotein cross-linking experiments [ 14]. It is now evident that the anticodon stem of P sitebound tRNA is positioned on the head of the 30S subunit, just above the cleft (fig 4). The proposed location has been documented by the fact that photoreactive analogs of the anticodon arm of yeast tRNAPhe containing 2- or 8-azidoadenosine at position 43 establish short range cross-links to proteins S13 and, to a lesser extent, S19 [41]. This placement is also in accord with the labeling of protein.S19 by E coil tRNAPhe derivatives with 14- and 20-A probes attached to position 47 [5]. Finally, several UVinduced cross-links from E coli tRNA phe, such as that between U60 in the T loop and protein $9 [54, 59], fit well with our new model. $9 maps close to S 13 at the top of the 30S subunit interface [6], and it has been cross-linked to protein L2 in the 50S subunit by treatment of 7 0 S ribosomes with bifunctional reagents [14]. A cross-link between G44 in the variable loop and protein L5 [54] can also be rationalized with the current model if we assume that this protein, located on the central protuberance [6], extends to the edge of the interface cavity. Protein L5 has, in addition, been labeled by E coli derivatized at position 47 [5]. In this case, the cross-link can be accounted for without the foregoing assumption because the long probes used in these experiments can easily reach the central protuberance. Much less cross-linking data are available for tRNA bound to the 30S-subunit portion of the A site. The relative positions of the A and P sites are determined, however, by the 3' ---> 5' orientation of the mRNA across the 30S subunit interface (fig 2), and from conclusions reached in our consideration of the 50S subunit (fig 3a). As depicted in the interface view of the 30S subunit (fig 4), the A-site tRNA is placed to the left of the P-site tRNA. This is in general agreement both with the labeling of protein S19 by E coli tRNA phe derivatized with a 9-A probe at nucleotide s4U8 [60] and with the UV-induced cross-link between protein S 10 and nucleotide A9 of unmodified E coli tRNAphe. Of all the cross-links listed in table I, only the labeling of protein $5 from position 21 of P-site tRNA [59]
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cannot be explained by our model, as this protein has been located on the solvent side of the 30S subunit by immune electron microscopy [6]. It is possible that protein $5 was not correctly identified, a~; this protein has frequently been cited as a cross-linking partner in studies of UV-induced tRNA-ribosome bonds in which the identification procedure involved iodination of isolated protein-tRNA complexes in conjunction with 2-dimensional polyacrylamide gel electrophoresis [25, 59]. Protein $5 was not found to be crosslinked to tRNA in the latest study of this series [54], in which protein identification was accomplished immunologically, without prior iodination. Although the E and R sites have until recently remained open to conjecture, the location of the E site is now quite clear and that of the R site can be defined from circumstantial evidence. From alterations in the susceptibility of bases in the 23S rRNA to chemical modification, Moazed and Noller [61] concluded that the 3' end of E site-bound tRNA contacts the 50S subunit interface somewhere in the valley that separates the L I ridge from the central protuberance. We now have evidence that both yeast and E coli tRNA~o containing 2-azidoadenosine as the 3'-terminal residue label protein L33, (J Wower, P Scheffer,W Wintermeyer and RA Zimmermann, unpublished results). It is noteworthy in this regard that protein L33 has been cross-linked to C2427 in the 23S rRNA [12], close to the msU residue at position 2394 which was found to be strongly protected from chemical modification by E site-bound tRNA [61]. Given the position of L33 on the 50S subunit [9], there can be little doubt that the E site is located between LI and L27 to the left of the P site (fig 3b). As aminoacyl-tRNA binds to the R site in a ternary complex with EF-Tu and GTP, the position of the R site on the ribosome should correspond to the EF-Tu binding site. This site was localized to the base of the L7/LI2 stalk on the interface of the 50S subunit [56] and ito the concave edge of the external surface of the 30Sisubunit, not far from S10 [62], by immune electron microscopy. The anticodon of R site-bound tRNA must occupy the same position as the anticodon of A site-bound tRNA. Because puromycin can still react with peptidyl-tRNA when aminoacyl=tRNA is bound to the ribosome in a ternary complex with EFTu and GTP [63, 64], the 3' end of the R site tRNA must be at some distance from the peptidyl transferase center (fig 3b).
Conclusion The effective use of chemical or photochemical crosslinking to map the locations of tRNA binding sites on the ribosome depends ultimately on a detailed knowledge of ribosome structure. Recent progress in this area, marked by the construction of the first 3-dimensional models of the ribosomal subunits that specify the disposition of both their RNA and protein consti-
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J Wower, RA Zimmermann
tuents, prompted us to review the accumulated crosslinking data to refine our working model of the tRNAribosome complex. We found that only a fraction of these data were defined precisely enough to be useful in locating the tRNA binding sites. Moreover, it became apparent that none of the published data on models of the tRNA-ribosome complex are fully consistent with the relevant cross-linking results. We have therefore advanced a new model which accounts for nearly all of the pertinent evidence. The placement of the aminoacyl arms of the A- and P-site tRNAs in the interface cavity of the 50S subunit, and the occurrence of codon-anticodon interaction within the cleft of the 30S subunit, indicate that specific topographical features of the ribosome may be crucial to its function in protein biosynthesis. Acknowledgments We are grateful to our colleagues, L Sylvers, Y Xing, P Scheffer, W Wintermeyer and S Hixson, for their contributions to the work presented here. We also acknowledge the support of grant GM 22807 from the National Institutes of Health.
References 1 Cooperman BS (1980) In: Ribosomes: Their Structure, Function and Genetics (Chambliss G, Craven GR, Davies J, Davis K, Kahan L, Nomura M, eds) University Park Press, Baltimore, 531-554 2 CoopermanBS (1987) Pharmacol Ther 34, 271-302 3 Cooperman BS (1988) Methods Enzymol 164, 341-361 4 Ofengand J (1980) In: Ribosomes: Their Structure, Function, and Genetics (Chambliss G, Craven GR, Davies J, Davis K, Kahan L, Nomura M, eds) University Park Press, Baltimore, 497-529 5 Ofengand J, Ciesiolka J, Denman R, Nurse K (1986) In: Structure, Function and Genetics of Ribosomes (Hardesty B, Kramer G, eds) Springer, NY, 473-494 6 St6ffler G, St6ffler-Meilicke M (1986) In: Structure, Function, and Genetics of Ribosomes (Hardesty B, Kramer G, eds) Springer, NY, 28-46 7 Oakes M, Henderson E, Scheinman A, Clark M, Lake JA (1986) In: Structure, Function and Genetics of Ribosomes (Hardesty B, Kramer G, eds) Springer, NY, 47-67 8 Capel MS, Engehnan DM, Freeborn BR, Kjeldgaard M, Langer JA, Ramakrishnan V, Schindler DG, Schneider DK, Schoenborn BP, Sillers IY, Yabuki S, Moore PB (1987) Science (Washington, DC) 238, 1403-1406 9 Waileczek J, Schiller D, St6ffler-Meilicke M, Brimacombe R, St6ffler G (1988) EMBO J 7, 3571-3576 10 Brimacombe R, Atmadja J, Stiege W, Schiller D (1988) JMolBiol 199, 115-136 11 Stern S, Weiser B, NoUer HF (1988) J Mol Biol 204, 447-48 ! 12 Mitchell P, Osswald M, Schueler D, Brimacombe R (1990) Nucleic Acids Res 18, 4325-4333 13 Watson JD (1964) Bull Soc Chim Bio146, 1399-1425 14 Traut RR, Tewari DS, Sommer A, Gavino GR, Olson HM, Glitz DG (1986) In: Structure, Function, and Genetics of Ribosomes (Hardesty B, Kramer G, eds) Springer, N'Y, 286--308
15 WettsteinFO, Noll H (1965)J Mol Biol 11, 35-53 16 Rheinberger H, Sternbach H, Nierhaus KH (1981) Proc Natl Acad Sci USA 78, 5310-5314 17 GrajevskajaRA, Ivanov YV, Saminsky EM (1982) Fur J Biocbem 128, 47-52 18 KirillovSV, Makarov EM, Semenkov Yu P (1983) FEBS Lett 157, 91-94 19 Lill R, Robertson JM, Wintermeyer W (1984) Biochemistry 23, 6710-6717 20 Geigenmiiller U, Nierhaus KH (1990) EMBO J 9, 4527--4533 21 Hardesty B, Culp W, McKeehan W (1969) Cold Spring Harb Syrup Quant Bio134, 331-345 22 LakeJA (1977) Proc NatlAcadSci USA 74, 1903-1907 23 Johnson AE, Fairclough RH, Cantor CR (1977) In: Nucleic Acid-Protein Recognition (Vogel HJ, ed) Academic Press, NY, 469-490 24 MoazedD, Noller HF (1989) Nature (Lond) 342, 142-148 25 Ahdurashidova GG, Baskayeva IO, Chernyi AA, Kazimir LB, Budowsky EI (1986) Eur J Biochem 159, 103-109 26 Budowsky EI, Abdurashidova GG (1989) Prog Nucleic Acid Res Mol Bio137, 1-65 27 Graifer DM, Babkina GT, Matasova NB, Vladimirov SN, Karpova GG, Vlasov VV (1989) Biochim Biophys Acta 1048, 245-256 28 Kruse TA, Siboska GE, Clark BFC (1982) Biochimie 64, 279-284 29 Cooperman BS, Dondon J, Finelli J, GrunbergManago M, Michelson AM (1977) FEBS Lett 76, 59--63 30 Riehl N, Remy P, Ebel JP, Ehresmann B (1982) Fur J Biochem 128, 427-433 31 RiehlN, Carbon P, Ehresmann B, Ebel JP (1984) Nucleic Acids Res 12, ~A.45--4453 32 Wower J, Hixson SS, Zimmermann RA (1988) Biochemistry 27, 8114--8121 33 Wower J, Hixson SS, Zimmermann RA (1989) Proc Natl Acad Sci USA 86, 5232-5236 34 Sylvers LA, Wower J, Hixson SS, Zimmermann RA (1988) FEBS Lett 245, 9-13 35 Walleczek J, Martin T, Redl B, StSffier-Meilicke M, St6ffier G (1989) Biochemistry 28, 4099--4105 36 Hackl W, St6ffler-Meilicke M, St6ffler G (1988) FEBS Lett 233, 119-123 37 Steiner G, Kuechler E, Barta A (1988) EMBO J 7, 3949-3955 38 Stiege W, Glotz C, Brimacombe R (1983) Nucleic Acids Res 11, 1687-1706 39 Prince JB, Taylor BH, Thurlow DL, Ofengand J, Zimmermann RA (1982) Proc Nail Acad Sci USA 79, 5450-5454 40 Gornicki P, Nurse K, Hellmann W, Boublik M, Ofengand J (1984) J Biol Chem 259, 10493-10498 41 Wower J, Malloy TA, Hixson SS, Zimmermann RA (1990) Biochim Biophys Acta 1050, 38--44 42 Stade K, Rinke-Appel J, Brimacombe R (1989) Nucleic Acids Res 17, 9889-9908 43 Oison HM, Lasater LS, Cann PA, Glitz DG (1988) J Bioi Chem 263, 15196-15204 44 Lake JA (1979) In: Transfer RNA: Structure, Properties and Recognition (Schimmel PR, $611 D, Abelson JN, eds) Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 393-411 45 Bollen A, Kahan L, Heimark RL, Cozzone A, Traut RR, Hershey JWB (1975) J Biol Chem 250, 4310--4314 46 Heimark RL, Kahan L, Johnston K, Hershey JWB, Traut RR (1976) J Mol Biol 105,219-230
A model of the tRNA-ribosome complex 47
Olson HM, Grant PG, Cooperman BS, Glitz DG (1982) J
Biol Chem 257, 2649-2656 48 Spirin AS (1983) FEBS Lett 156, 217-221
49
Ofengand J, Lin FL, Hsu L, Boublik M (1981) In:
55 56
Molecular Approaches to Gene and Protein Structure (Siddiqui, MAQ et al, eds) Academic Press, Inc, NY,
57
1-31 50 Gold L (1988) Annu Rev Biochem 57, 199-233 51 Moazed D, Noller I-IF (1986) Cell 47, 985-994 52 Noller HF, Moazed D, Stem S, Powers T, Allen PN, Robertson JM, Weiser B, Triman K (1990) In: The Ribosome: Structure, Function and Evolution (Hill WE, Dahlberg A, Garrett RA, Moore PB, Sehlessinger D, Warner JR, eds) Am Soe Micmbiol Washington, DC, 73-92 53 Wagenkneeht T, Carazo JM, Radermaeher M, Frank J (1989) Biophys J 55,455-464 54 Abdurashidova GG, Tsvetkova EA, Budowky E1 (1990) FEBS Lett 269, 398--401
58 59 60 61 62 63 64
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Hoppe W, Oettl H, Tietz HR (1986) J Mol Biol 192, 291-322 Girshovich AS, Bochkareva ES, Vasiliev VD (1986) FEBS Lett 197,. 192-198 Kao TH, Miller DL, Abo M, Ofengand J (1983) J Mol Biol 166, 383--405 Podkowinski J, Gomicki P (1989) Nucleic Acids Res 17, 8767-8782 Abdurashidova GG, Tsvetkova EA, Budowsky EI (1989) FEBS Lett 243, 299-302 Lin FL, Kahan L, Ofengand J (1984) J Moi Biol 172, 77-86 Moazed D, Noller HF (1989) Cell 57, 585-597 Langer J, Lake JA (1986) J Mol Biol 187, 617621 Lucas-Lenard J, Tao P, Haenni A (1969) CoM Spring Harbor Syrup Quant Bio134, 455--462 Shorey RL, Ravel JM~ Shive W (1971) Arch Biochem Biophys 146, 110--117
© Soci6t6 franqaise de biochimie et biologie mol6culaire / Elsevier, Pads
961
A consonant model of the tRNA-ribosome complex during the elongation cycle of translation J Wower, RA Zimmermann* Department of Biochemistry, University of Massachusetts, Amherst, Massachusetts 01003, USA
(Received 26 January 1991; accepted 13 March 1991)
Summary - - Chemical and photochemical affinity techniques have been used extensively to determine the positions of the tRNA binding sites on the Escherichia coil ribosome. Recent advances in our understanding of ribosome structure and function prompted us to critically review the data that have accumulated on tRNA-ribosome cross-links. As a result, we propose a new model of the tRNAribosome complex that accounts for nearly all of the pertinent evidence. affinity labeling / cross-linking techniques / tRNA-ribosome complex / peptidyi transferase center / decoding site
Introduction
tRNA binding sites on the ribosome
During the last 20 years, numerous cross-linking studies designed to locate the tRNA binding sites on the Escherichia coli ribosome have been carried out [1-5]. Over the same period, our understanding of the structure and function of the ribosome has advanced significantly. Almost all of the ribosomal proteins have now been mapped on the surface of the ribosomal subunits by immune electron microscopy [6, 7]. In addition, the 3-dimensional organization of the proteins in the 30S subunit has been defined by neutron scattering [8]. While similar studies on the 50S subunit are not yet complete, important insights into the spatial relationships among 50S-subunit proteins have been gained by combining proteinprotein cross-linking results with those derived from immune electron microscopy [6, 9]. Furthermore, the development of techniques for studying the intra- and intermolecular interactions of large RNAs has le6 to the construction of rather detailed 3-dimensional models of the 16S rRNA [10, 11] and a portion of the 23S rRNA [12], together with their associated proteins. These advances in our knowledge of ribosome structure prompted us to re-examine the available data on tRNA-ribosome cross-linking. The result is a new model of the tRNA-ribosome complex that is consistent with most of the pertinent structural information.
In the original model of the translating ribosome prop,~sed by Watson [13], ribosomes were envisioned to have only two tRNA binding sites, a donor (D) site for the tRNA donating its peptidyl moiety to the growing polypeptide chain and an acceptor (A) site for the aminoacyl-tRNA accepting this moiety during formation of the peptide bond. The donor (D) and acceptor (A) sites are now generally known as the peptidyl (P) and aminoacyl (A) sites, respectively (fig l a). These two sites are functionally distinct, as only peptidyl-tPNA bound to the P site is able to react with puromycin, an antibiotic which acts as an analog of the terminal aminoacyl-adenosine portion of A sitebound aminoacyl-tRNA [14]. This two-site model was later modified to include two more tRNA binding sites (fig lb). One of them, initially proposed by Wettstein and Noll [15] and now called the exit (E) site, receives deacylated tRNA from the P site prior to its release from the ribosome. Convincing evidence for the existence of this site has been provided by several groups [ 16-19] and its role in maintaining translational accuracy has recently been substantiated [20]. The presence of another site, the recognition (R) site, to which the aminoacyl-tRNA.EF-Tu.GTP ternary complex binds, was originally proposed by Hardesty et al [21] and later incorporated into the models of Lake [22] and Johnson et al [23]. Recent studies by Noller et al have raised the possibility that there are two intermediate states, designated P/E and A/P to emphasize their hybrid character
*Correspondence and reprints
962
J Wower, RA Zimmermann
(a)
(b)
(c)
Fig 1. Evolution of the concept of tRNA binding sites on the ribosome, a) The two-site model of Watson [13]; b) the tbur-site model; c) the four-site model with intermediate states added. A, aminoacyl site; P, peptidyi site; R, recognition site; E, exit site. The A/P and P/E sites of Moazed and Noller [24] correspond to At, and Ph sites of Abdurashidova et al [251, respectively (see text).
124], which are occupied by tRNAs immediately after peptide bond formation (fig l c). In the P/E site, the anticodon of the tRNA is stabilized in the P site through base pairing with the mRNA codon, while the 3' end is located in the 50S-subunit E site. In a similar vein, the anticodon of tRNA in the A/P site is thought to remain in the A site, while the 3' end, carrying the peptidyl moiety, has been transferred to the P site. According to this view, the R, A, P and E sites are designated the Aft, A/A, P/P and E sites, respectively. The six-state model of Moazed and Noller [241 closely resembles a model proposed three years earlier by Budowsky et al [25]. Here, the R, A, P and E sites are called Ar, A~, Pt (or Pr) and S, respectively. In addition to these four sites, two additional sites, Ab and Pb, correspond to the A/P and P/E sites. Given that the data concerning sites other than R, A, P and E come only from Noller's and Budowsky's laboratories, the designations proposed by these authors will be used in what follows to avoid confusion. Strategies f o r cross-linking tRNA to the ribosome
Cross-linking provides a straightforward means for !dentifying ribosomal components that are close to, or m contact with tRNA bound to functional ribosomal complexes. Two general approaches have been developed in the last two decades. One approach, designed for cross-linking unmodified tRNAs, relies on the direct photolysis of tRNA-ribosome complexes by UV light [24]. Irradiation at 254 nm, however, generates random cross-links between tRNA and the ribosome, as well as spurious intraribosomal cross-links that inactivate the ribosomal particles. To avoid this,
some workers have induced photolysis by irradiation with UV light at wavelengths between 300--400 nm. In general, such an approach is useful only for tRNAs containing naturally photoreactive nucleosides since the four commonly occurring nucleosides exhibit relatively low photoreactivity at these wavelengths. Because of the scarcity of tRNAs with suitably reactive residues, most cross-linking studies have utilized a second approach in which tRNAs derivatized with chemically or photochemically reactive probes [3]. In most instances, the probes have been attached either to the a-amino group of aminoacyltRNA or to one of a number of positions in tRNA occupied by naturally occurring, modified bases. In one case, the guanosine residues of tRNA have been derivatized randomly [27]. The main drawback of this approach is that the long and bulky substituents which are frequently used can potentially interfere with the proper binding of tRNA to the ribosome. In addition, the cross-links that result from the use of such probes are necessarily at some distance from the actual tRNA binding site. In an effort to avoid the shortcomings of the two approaches described above, three new strategies have been developed. In one, flavin mononucleotide (FMN) irradiated at 350 nm was used as a photosensitizer; by generating singlet oxygen, superoxide and hydroxyl radicals, the activated FMN initiates free-radical processes leading to tRNA-ribosome cross-linking [28]. However, because photosensitization in itself leads to rapid inactivation of ribosomes [29], this method is not particularly advantageous for crosslinking studies. In the second strategy, cross-linking is performed with tRNA molecules in which cytidine residues have been chemically converted to 4-thiouridine (s4U) [30, 31 ]. As this nucleoside is photoreactive at 335 nm, the thiolated tRNAs can be directly cross-linked to ribosomes without affecting their structure or function. While this approach has great potential for analyzing ribosomal components in the neighborhood of cytidine residues in ribosome-bound tRNA, suitable means must be developed to separate aminoacyltRNA from the background of non-aminoacylated tRNA and to determine the identity of the cross-linked residue(s) after photolysis. Over the past few years, we have developed a third strategy which entails the introduction of photoreactive azidonucleosides at defined positions in the tRNA molecule [32-34]. These nucleoside analogs, such as 2-azidoadenosine and 8-azidoadenosine, can in principle be inserted at any position as long as the substitution does not interfere with the function of the tRNA molecule. Cross-linking of the azidonucleosidecontaining tRNAs to ribosomes is achieved by irradiation of noncovalent tRNA-mRNA-ribosome complexes with light of 300 nm, a wavelength which
A model of the tRNA-ribosome complex neither generates spurious cross-links nor disrupts the structure or function of the ribosome. Since the photochemical reaction leads to the formation of very short cross-links (2-4 A), only those ribosomal components in close proximity to the photoreactive nucleoside become covalently attached.
Identification of ribosomal components at the peptidyi transferase center and in the decoding domain The majority of cross-linking experiments have been carried out with aminoacyl-tRNAs derivatized on their aminoacyi moiety in order to identify ribosomal component(s) at the peptidyl transferase center of the ribosome. These probes react exclusively with 50S ribosomal subunits. Among the ribosomal proteins most consistently labeled are L2, LI5, LI6 and L27 [5]. Significantly, the close proximity of these proteins within the 50S subunit has been demonstrated by inter-protein cross-linking [14, 35]. Moreover, it has been shown by immune electron microscopy [6, 36] that proteins L2, L15 and L27 are clustered on the interface of the 50S subunit in the valley between the L1 ridge and the central protuberance (fig 2a). Although the 23S rRNA was labeled by various derivatized aminoacyl-tRNAs, sites of cross-linking have only been precisely identified for 3-(4'-benzoylphenyl)propionyl-Phe-tRNAPhe [37]. In the latter case, nucleotides A2451 and C2452 were cross-linked by P site-bound tRNA while nucleotides U2584 and U2585 were crossqinked to the tRNA when it was located in the A site. A rain, :"reaction with the AUGU sequence spanning positions 2503-2506 was observed when 3(4'-bcnzoylph,:,yl)propionyl-Phe-tRNA was in either
3'
(o)
(b)
Fig 2. Models of the 30S and 50S ribosomal subunits of E coli. a) 50S subunit; the cluster of proteins most frequently labeled by aminoacyl-tRNAs derivatized on the aminoacyl moiety is cross-hatched; numbers represent positions of 50S subunit patterns as in [6, 36]. b) 30S subunit; orientation of the mRNA according to Olson et al [43]. 1400, the location of nucleotide C1400 of the 16S rRNA [40].
963
site. Interestingly, although the labeled nucleotides are not adjacent to each other in the primary structure of the 23S rRNA, they all reside in the same portion of the secondary structure which is referred to as the central loop of domain V and is often identified with the peptidyl transferase center. This structure is expected to be in close proximity to protein L2 in the 50S ribosomal subunit since nucleotide U1782, within the binding site for protein L2, has been cross-linked to nucleotide U2609, within the central loop [38]. In addition, short range cross-links have been established to proteins L2, L15, LI6 and L27, as well as to nucleotide G1945 of the 23S rRNA from yeast tRNA phe variants containing azidoadenosine at positions 76 and 73, one or four bases removed from the aminoacyl group, respectively [33]. While the cross-linking studies carried out with aminoacyl-tRNAs derivatized on their aminoacyl moiety or with azidonucleosides incorporated at or near their 3' terminus have not identified a unique ribosomal component or structure that might correspond to peptidyl transferase, they do indicate that the aminoacyl arms of the L-shaped tRNAs at both the A and P sites interact with a limited area of the 50S subunit defined by proteins L2, LI5, LI6 and L27, and portions of domains IV and V of the 23S rRNA (fig 2a). Since mRNA binds exclusively to the 30S ribosomal subunit, the decoding domain - the site or sites at which the tRNA anticodons interact with the mRNA codons - must be located on this subunit. To define this domain, affinity-labeling experiments have been performed with reactive derivatives of both tRNA and mRNA [2, 3]. In one case, photochemical cross-linking of E coil tRNA val to the 30S-subunit P site was found to be mediated by the 5' anticodon base of the tRNA and residue C1400 of the 16S rRNA [39]. Investigation of this complex by immune electron microscopy [40] demonstrated that the site of codon-anticodon interaction lies in the cleft between the head and the platform of the 30S subunit (fig 2b). Cross-links from azidoadenosines at positions 37 in the anticodon loop to protein $7, and at position 43 in the anticodon stem to proteins S13 and S19 are consistent with this localization ([41]; LA Sylvers, J Wower and RA Zimmermann, unpublished data). When mRNA analogs with affinity probes attached to their 5' or 3' termini were used to elucidate mRNAribosome interactions, several proteins, covering a large fraction of the 30S subunit surface, were labeled [3]. Consequently, these results did not permit a clear definition of the mRNA-binding region. More recent studies, employing mRNA analogs with reactive nucleosides at internal positions, have shown that proteins $7, $21 and a segment of the 16S rRNA very close to its 3' terminus are within the decoding domain [42].
964
J Wower, RA Zimmermann
A matter of critical importance for understanding the way in which tRNA molecules are positioned on the 30S subunit is the orientation of the mRNA. Two independent lines of evidence demonstrate that the mRNA crosses the cleft in a 3' to 5' direction, when viewed from the 30S subunit interface (fig 2b). In the first, mRNA analogs with antigenic substituents at either the 5' or 3' end were bound to the 30S particle, allowed to interact with the appropriate antibodies, and analyzed by immune electron microscopy [43]. In the second, the path of the mRNA was inferred from cross-links to 16S rRNA and 30S subunit proteins from synthetic mRNA analogs containing, at three different positions, a single thiouridine residue derivatized with 4-azidophenacylbromide [42]. P site-bound tRNA must be positioned so that its anticodon is anchored by the mRNA in the cleft of the 30S subunit while its aminoacyl stem is directed toward the 50S subunit. It follows that the tRNA in the A site should be similarly positioned as both the 3' termini and the anticodons of the two tRNAs must be closely juxtaposed. Because mRNA is translated in a 5'-->3' direction, the A-site tRNA must be to the left, and the Psite tRNA to the right, of the interface surface of the 30S subunit (fig 4). While it is now generally agreed that the peptidyl transferase center is located between the central protuberance and the L1 ridge of the 50S subunit and that the decoding domain is situated between the head and the platform of the 30S subunit, little is known about other contacts between tRNA and the ribosome. Models of tRNA.mRNA.ribosome complex To aid in interpreting the data on the location of the ribosomal binding sites for tRNA, several models of the tRNA-ribosome complex have been proposed in the course of the last 20 years. These will be briefly discussed below, in historical order.
INrERFACE
CAVITY
(a)
(b)
Fig 3. Model for the arrangement of tRNAs on the 50S subunit of the E coli r~bosome, a) Peptidyl (P) and aminoacyi (A) sites. (b) Exit (E), peptidyl (P) and recognition (R) sites. Numbers indicate locations of 50S subunit proteins according to [6, 36].
|
3'
Fig 4. Model for the arrangement of tRNAs in the aminoacyl (A) and peptidyl (P) sites of the 30S subunit of the E coil ribosome. Numbers indicate locations of 30S subunit proteins according to [6]. One of the first models, advanced by Lake [22], proposed locations for three tRNA molecules on the 30S subunit, at the R, A and P sites. This model was later extended to include the 50S subunit [44]. The sites of interaction of the tRNAs on the ribosome were deduced from immune electron-microscopic mapping of the ribosomal proteins and from information available at the time about the function of these proteins. In Lake's model, the R site-bound tRNA is located on the solvent side of the 30S subunit in such a way that its anticodon is anchored at the solvent end of the cleft by anticodon-codon interaction with mRNA and its Lshaped body is adjacent to proteins $5, $8, S10, S12 and S14. The anticodon of the A site-bound tRNA must be at the same location as that of the R sitebound tRNA. Based on the location of proteins crosslinked to initiation factors IF2 [45] and IF3 [46], which guide tRNA~ et to the 30S subunit, P site-bound tRNA was placed within the cleft. The relative positions of the A and P sites in this model, however, would require a 5'-+3' orientation of the mRNA across the small-subunit interface. Identification of protein S14 as a major target for puromycin cross-linking prompted Olson et al [47] to propose a slightly different model for tRNA at the A site of the 70S ribosome. While the position of the Asite tRNA in this version is very similar to that in Lake's model, the aminoacyl acceptor end of the tRNA interacts with the head rather than the platform of the 30S particle on the subunit interface. Another model for the arrangement of tRNA molecules on the ribosome was suggested by Spirin [48]. In this scheme, the anticodons of the tRNAs bound to the A and P sites are located on the solvent side of the 30S subunit, in the groove separating its head and body, and their aminoacyl acceptor ends lie in a valley, just below the central protuberance, on the
A model of the tRNA-ribosomecomplex interface side of the 50S subunit. The A site is formed in part by the portion of the 30S subunit head that encompasses proteins S14 and S 19, and in part by the central protuberance of the 50S subunit. The P site is located on the surface of the 30S subunit in the neighborhood of protein $5 and the surface of the 50S subunit at the base of the L7/L 12 stalk. Cross-linking of yeast tRNAphe with probes attached to the central portion of the L-shaped molecule led to a fourth model of the ribosomal A and P sites [5, 49]. In this model, the anticodons of both tRNAs are in the cleft while their aminoacyl ends point toward the head of the 30S subunit. The tRNAs are oriented on the 30S subunit in such a way that, after binding to the 50S subunit, the P-site tRNA is positioned in the valley between the LI ridge and the central protuberance, while the A-site tRNA is closer to the L I ridge. Here again, the locationa of the Aand P-site tRNAs do not accord with me mRNA orientation derived from immune electron microscopy and cross-linking studies [42, 43]. In the model of Gold [50], the mRNA and tRNAs are placed on opposite sides of the 30S ribosomal subunit" the mRNA binds to an open 'trough' carved into the solvent side, and the tRNAs are located in the space between the ribosomal subunits. In order to interact with mRNA, the tRNA anticodons protrude into the trough through 'slots' in the 30S subunit. The 3' ends of A- and P-site tRNAs project toward the 50S subunit to contact the peptidyl transferase center while the E-site tRNA is placed in the vicinity of the L1 ridge. The relative positions of the A, P and E sites on the ribosome are in agreement with those implied by the 3'--->5'orientation of the mRNA on the 30S subunit interface. A model recently proposed by this laboratory was based largely on photoaffinity labeling studies with azidonucleoside-substituted derivatives of yeast tRNAphe [33]. Ip this depiction, the aminoacyl arms of both A- and P-site tRNAs are directed toward the peptidyl transferase center on the interface surface of the 50S subunit, while the anticodons interact with mRNA in the cleft of the 30S subunit. The A site is located to the left of the P site when viewed from the 30S subunit interface, consistent with the preferred orientation of the mRNA. A model quite similar in concept to the pre.ceding was derived from a very different expenmental approach - an analysis of alterations in the chemical reactivity of rRNA bases elicited by tRNA bound to the A, P and E sites. First advanced on the basis of results for the 30S subunit [ 11], this model was later extended to the 50S subunit by the inclusion of chemical modification data from the 23S rRNA [52]. Because the 3' terminus of E site-bound tRNA was found to protect several bases within the binding site for protein L l, these authors inferred that the E site is located between the L1 ridge and the central protuberance.
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Using 3-dimensional image reconstruction in conjunction with electron microscopy, Wagenknecht et al [53] have recently identified a large cavity, the 'interface canyon', just below the central protuberance on the interface of the 50S subunit. These authors suggest that the interface canyon could accommodate the aminoacyl stems of the A and P site-bound tRNAs. According to this model, the anticodons of the tRNAs are positioned on the surface of the 30S subunit as suggested earlier by Spirin [48]. A new model of the tRNA-ribosome complex
In this section, we develop a model for the arrangement of tRNA binding sites on the ribosome that is based mainly on tRNA-ribosome cross-linking results. The data on tRNA-ribosome cross-linking can be divided into two main categories. The first category derives from experiments that provide information not only about the labeled ribosomal components but also about the site in the tRNA molecule from which the cross-link occurred (table I). The second category only specifies the ribosomal components that were labeled [25, 27, 31]. Further consideration of the arrangement of tRNAs on the ribosome will only be based on data from the first category, as the latter are incomplete from a topographical point of view. Moreover, we have included, for the purposes of this discussion, only those experiments in which the binding site occupied by the cross-linked tRNA was satisfactorily defined. As indicated earlier, the available data favor models that: a) place the anticodons of A and P site-bound tRNAs in the cleft of the 30S ribosomal subunit; b) orient the aminoacyl arms of these tRNAs toward the L2-L15-L16-L27 protein cluster on the interface surface of the 50S subunit; and c) predict a relative arrangement of the A- and P-site tRNAs that is consistent with the 3'--->5' directionality of the mRNA across the interface surface of the 30S subunit. Although the models proposed by Wower et al [33] and Noller et al [52] fulfil these three criteria, they do not always account for the most recent cross-linking results listed in table I. For example, the cross-linking of nucleotides located both at the 3' terminus (positions 73 and 76) and within the central region (positions 17, 18, 56 and 59) of the P site-bound tRNA to proteins L2 and L27 via short-range bonds [32, 33, 54], cannot be accounted for by any model of the tRNA-ribosome complex suggested so far. To explain these data, one has to accept the notion that the aminoacyl acceptor arm of the P-site tRNA, and perforce the A-site tRNA, are immersed in a cavity on the interface side of the 50S subunit that is bounded, at least in part, by proteins L2 and L27 (fig 3a). As indicated earlier, the presence of such a cavity in the 50S subunit has already been inferred from electron microscopy [53, 55].
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Table I. tRNA-ribosome cross-links Ribosomal site
tRNA
Positwn in tRNA
Probe
Labeled component
Ref
p
tRNAphe
A76
2-4 A (azide)
L27, LI5, LI6 G 1945/23S RNA L27
[33]
2--4/~, (azide)
L27 L27, L2
[32]
14-20 ]k (300 nm)
S 19, L5, L27
[5]
G43
2-4 A (azide)
SI3, S19
[411a
t6A37
17 A 24 A
$7, LI, L27, L33 $7
[581
Y37
2-4 A (azide)
$7
cmoSU34
3-4 A (300 nm)
C 1400/16S RNA
[391
U60 u45 A21 U60 C56 Cl7 U60 C56 G44 C17
2-4 A (254 nm)
S9 $7 $5 $9 L27 L2 S9 L27 L5 L2
[59]
U59 A26 G 18 A9
2-4 A, (254 nm)
L2 $7 L27 Sl0
[54]
E coli
tRNAPhe
s4U8
9/~
S 19
[60]
A76
2-4 A, (azide)
L33
yeast
A73
p
tRNAPhe yeast
A76 A73
P
tRNAphe
acp3U47
E coli
p P P P
tRNAPhea yeast tRNA.Met yeast tRNAphe yeast tRNAIval E coil
P
tRNAphe E coli
Pt
tRNAPhe E coli
Pb
tRNAPhe E coli
Ab
A
tRNAphe
2-4/~ (254 nm) 2-4 A (254 nm)
b
[541 [54]
E coli
E
tRNAphe E coli
aAnticodon arm (residues 28-43); bLA Sylvers, J Wower, RA Zimmermann, unpublished results; cJ Wower, P Scheffer, W Wintermeyer., RA Zimmermann, unpublished result. Placement of the acceptor arms of the A- and P-site tRNAs within the interface cavity of the 50S subunit also provides a simple explanation for the persistent labeling of proteins L2 and L27 by or-amino derivatives of aminoacyl-tRNAs [5]. Moreover, this proposition suggests a way in which the ribosome could facilitate formation of the peptide bond. As no single ribosomal component exhibits high affinity toward the 3' terminus of the tRNA molecule, it is possible that the interface cavity, a feature whose structure is attributable to a number of components, guides the 3' ends of tRNAs to the peptidyl transferase center.
In our model, the 50S-subunit portion of the A site has been positioned to the right of the P site when viewed from the interface side (fig 3a). Placement of the A-site tRNA near protein LI 1 is consistent with the location of the EF-Tu binding site at the base of the L7/LI2 stalk [56]. It is also in accordance with the finding that E coli tRNAPhe, cross-linked via position 47 to EF-Tu, can bind to the A site when the P site is occupied by aminoacylated tRNA [57]. The disposition of the P site on the 30S subunit is now delineated by a number of cross-links (fig 4). As cmosU34 of E coli tRNAyal can form a cyclobutane
A model of the tRNA-ribosomecomplex dimer with C1400 of the 16S rRNA, the tRNA anticodon must lie within 3--4 A of the rRNA [39]. As mentioned above, the site of cross-linking has been mapped to the cleft of the 30S subunit [40]. The immediate surroundings of the anticodon loop have been further defined by a short-range cross-link from 2-azidoadenosine at position 37 of yeast tRNAphe to protein $7 (LA Sylvers, J Wower and RA Zimmermann, unpublished data). $7 is also the predominant protein labeled by yeast tRNA Met derivatized at position 37 with 17- and 24-A, probes [58]. Interestingly, the 17-A probe also reacts with protein L I in the 50S subunit. This result is consistent with the proximity of $7 and LI in the 70S ribosome inferred from proteinprotein cross-linking experiments [ 14]. It is now evident that the anticodon stem of P sitebound tRNA is positioned on the head of the 30S subunit, just above the cleft (fig 4). The proposed location has been documented by the fact that photoreactive analogs of the anticodon arm of yeast tRNAPhe containing 2- or 8-azidoadenosine at position 43 establish short range cross-links to proteins S13 and, to a lesser extent, S19 [41]. This placement is also in accord with the labeling of protein.S19 by E coil tRNAPhe derivatives with 14- and 20-A probes attached to position 47 [5]. Finally, several UVinduced cross-links from E coli tRNA phe, such as that between U60 in the T loop and protein $9 [54, 59], fit well with our new model. $9 maps close to S 13 at the top of the 30S subunit interface [6], and it has been cross-linked to protein L2 in the 50S subunit by treatment of 7 0 S ribosomes with bifunctional reagents [14]. A cross-link between G44 in the variable loop and protein L5 [54] can also be rationalized with the current model if we assume that this protein, located on the central protuberance [6], extends to the edge of the interface cavity. Protein L5 has, in addition, been labeled by E coli derivatized at position 47 [5]. In this case, the cross-link can be accounted for without the foregoing assumption because the long probes used in these experiments can easily reach the central protuberance. Much less cross-linking data are available for tRNA bound to the 30S-subunit portion of the A site. The relative positions of the A and P sites are determined, however, by the 3' ---> 5' orientation of the mRNA across the 30S subunit interface (fig 2), and from conclusions reached in our consideration of the 50S subunit (fig 3a). As depicted in the interface view of the 30S subunit (fig 4), the A-site tRNA is placed to the left of the P-site tRNA. This is in general agreement both with the labeling of protein S19 by E coli tRNA phe derivatized with a 9-A probe at nucleotide s4U8 [60] and with the UV-induced cross-link between protein S 10 and nucleotide A9 of unmodified E coli tRNAphe. Of all the cross-links listed in table I, only the labeling of protein $5 from position 21 of P-site tRNA [59]
967
cannot be explained by our model, as this protein has been located on the solvent side of the 30S subunit by immune electron microscopy [6]. It is possible that protein $5 was not correctly identified, a~; this protein has frequently been cited as a cross-linking partner in studies of UV-induced tRNA-ribosome bonds in which the identification procedure involved iodination of isolated protein-tRNA complexes in conjunction with 2-dimensional polyacrylamide gel electrophoresis [25, 59]. Protein $5 was not found to be crosslinked to tRNA in the latest study of this series [54], in which protein identification was accomplished immunologically, without prior iodination. Although the E and R sites have until recently remained open to conjecture, the location of the E site is now quite clear and that of the R site can be defined from circumstantial evidence. From alterations in the susceptibility of bases in the 23S rRNA to chemical modification, Moazed and Noller [61] concluded that the 3' end of E site-bound tRNA contacts the 50S subunit interface somewhere in the valley that separates the L I ridge from the central protuberance. We now have evidence that both yeast and E coli tRNA~o containing 2-azidoadenosine as the 3'-terminal residue label protein L33, (J Wower, P Scheffer,W Wintermeyer and RA Zimmermann, unpublished results). It is noteworthy in this regard that protein L33 has been cross-linked to C2427 in the 23S rRNA [12], close to the msU residue at position 2394 which was found to be strongly protected from chemical modification by E site-bound tRNA [61]. Given the position of L33 on the 50S subunit [9], there can be little doubt that the E site is located between LI and L27 to the left of the P site (fig 3b). As aminoacyl-tRNA binds to the R site in a ternary complex with EF-Tu and GTP, the position of the R site on the ribosome should correspond to the EF-Tu binding site. This site was localized to the base of the L7/LI2 stalk on the interface of the 50S subunit [56] and ito the concave edge of the external surface of the 30Sisubunit, not far from S10 [62], by immune electron microscopy. The anticodon of R site-bound tRNA must occupy the same position as the anticodon of A site-bound tRNA. Because puromycin can still react with peptidyl-tRNA when aminoacyl=tRNA is bound to the ribosome in a ternary complex with EFTu and GTP [63, 64], the 3' end of the R site tRNA must be at some distance from the peptidyl transferase center (fig 3b).
Conclusion The effective use of chemical or photochemical crosslinking to map the locations of tRNA binding sites on the ribosome depends ultimately on a detailed knowledge of ribosome structure. Recent progress in this area, marked by the construction of the first 3-dimensional models of the ribosomal subunits that specify the disposition of both their RNA and protein consti-
968
J Wower, RA Zimmermann
tuents, prompted us to review the accumulated crosslinking data to refine our working model of the tRNAribosome complex. We found that only a fraction of these data were defined precisely enough to be useful in locating the tRNA binding sites. Moreover, it became apparent that none of the published data on models of the tRNA-ribosome complex are fully consistent with the relevant cross-linking results. We have therefore advanced a new model which accounts for nearly all of the pertinent evidence. The placement of the aminoacyl arms of the A- and P-site tRNAs in the interface cavity of the 50S subunit, and the occurrence of codon-anticodon interaction within the cleft of the 30S subunit, indicate that specific topographical features of the ribosome may be crucial to its function in protein biosynthesis. Acknowledgments We are grateful to our colleagues, L Sylvers, Y Xing, P Scheffer, W Wintermeyer and S Hixson, for their contributions to the work presented here. We also acknowledge the support of grant GM 22807 from the National Institutes of Health.
References 1 Cooperman BS (1980) In: Ribosomes: Their Structure, Function and Genetics (Chambliss G, Craven GR, Davies J, Davis K, Kahan L, Nomura M, eds) University Park Press, Baltimore, 531-554 2 CoopermanBS (1987) Pharmacol Ther 34, 271-302 3 Cooperman BS (1988) Methods Enzymol 164, 341-361 4 Ofengand J (1980) In: Ribosomes: Their Structure, Function, and Genetics (Chambliss G, Craven GR, Davies J, Davis K, Kahan L, Nomura M, eds) University Park Press, Baltimore, 497-529 5 Ofengand J, Ciesiolka J, Denman R, Nurse K (1986) In: Structure, Function and Genetics of Ribosomes (Hardesty B, Kramer G, eds) Springer, NY, 473-494 6 St6ffler G, St6ffler-Meilicke M (1986) In: Structure, Function, and Genetics of Ribosomes (Hardesty B, Kramer G, eds) Springer, NY, 28-46 7 Oakes M, Henderson E, Scheinman A, Clark M, Lake JA (1986) In: Structure, Function and Genetics of Ribosomes (Hardesty B, Kramer G, eds) Springer, NY, 47-67 8 Capel MS, Engehnan DM, Freeborn BR, Kjeldgaard M, Langer JA, Ramakrishnan V, Schindler DG, Schneider DK, Schoenborn BP, Sillers IY, Yabuki S, Moore PB (1987) Science (Washington, DC) 238, 1403-1406 9 Waileczek J, Schiller D, St6ffler-Meilicke M, Brimacombe R, St6ffler G (1988) EMBO J 7, 3571-3576 10 Brimacombe R, Atmadja J, Stiege W, Schiller D (1988) JMolBiol 199, 115-136 11 Stern S, Weiser B, NoUer HF (1988) J Mol Biol 204, 447-48 ! 12 Mitchell P, Osswald M, Schueler D, Brimacombe R (1990) Nucleic Acids Res 18, 4325-4333 13 Watson JD (1964) Bull Soc Chim Bio146, 1399-1425 14 Traut RR, Tewari DS, Sommer A, Gavino GR, Olson HM, Glitz DG (1986) In: Structure, Function, and Genetics of Ribosomes (Hardesty B, Kramer G, eds) Springer, N'Y, 286--308
15 WettsteinFO, Noll H (1965)J Mol Biol 11, 35-53 16 Rheinberger H, Sternbach H, Nierhaus KH (1981) Proc Natl Acad Sci USA 78, 5310-5314 17 GrajevskajaRA, Ivanov YV, Saminsky EM (1982) Fur J Biocbem 128, 47-52 18 KirillovSV, Makarov EM, Semenkov Yu P (1983) FEBS Lett 157, 91-94 19 Lill R, Robertson JM, Wintermeyer W (1984) Biochemistry 23, 6710-6717 20 Geigenmiiller U, Nierhaus KH (1990) EMBO J 9, 4527--4533 21 Hardesty B, Culp W, McKeehan W (1969) Cold Spring Harb Syrup Quant Bio134, 331-345 22 LakeJA (1977) Proc NatlAcadSci USA 74, 1903-1907 23 Johnson AE, Fairclough RH, Cantor CR (1977) In: Nucleic Acid-Protein Recognition (Vogel HJ, ed) Academic Press, NY, 469-490 24 MoazedD, Noller HF (1989) Nature (Lond) 342, 142-148 25 Ahdurashidova GG, Baskayeva IO, Chernyi AA, Kazimir LB, Budowsky EI (1986) Eur J Biochem 159, 103-109 26 Budowsky EI, Abdurashidova GG (1989) Prog Nucleic Acid Res Mol Bio137, 1-65 27 Graifer DM, Babkina GT, Matasova NB, Vladimirov SN, Karpova GG, Vlasov VV (1989) Biochim Biophys Acta 1048, 245-256 28 Kruse TA, Siboska GE, Clark BFC (1982) Biochimie 64, 279-284 29 Cooperman BS, Dondon J, Finelli J, GrunbergManago M, Michelson AM (1977) FEBS Lett 76, 59--63 30 Riehl N, Remy P, Ebel JP, Ehresmann B (1982) Fur J Biochem 128, 427-433 31 RiehlN, Carbon P, Ehresmann B, Ebel JP (1984) Nucleic Acids Res 12, ~A.45--4453 32 Wower J, Hixson SS, Zimmermann RA (1988) Biochemistry 27, 8114--8121 33 Wower J, Hixson SS, Zimmermann RA (1989) Proc Natl Acad Sci USA 86, 5232-5236 34 Sylvers LA, Wower J, Hixson SS, Zimmermann RA (1988) FEBS Lett 245, 9-13 35 Walleczek J, Martin T, Redl B, StSffier-Meilicke M, St6ffier G (1989) Biochemistry 28, 4099--4105 36 Hackl W, St6ffler-Meilicke M, St6ffler G (1988) FEBS Lett 233, 119-123 37 Steiner G, Kuechler E, Barta A (1988) EMBO J 7, 3949-3955 38 Stiege W, Glotz C, Brimacombe R (1983) Nucleic Acids Res 11, 1687-1706 39 Prince JB, Taylor BH, Thurlow DL, Ofengand J, Zimmermann RA (1982) Proc Nail Acad Sci USA 79, 5450-5454 40 Gornicki P, Nurse K, Hellmann W, Boublik M, Ofengand J (1984) J Biol Chem 259, 10493-10498 41 Wower J, Malloy TA, Hixson SS, Zimmermann RA (1990) Biochim Biophys Acta 1050, 38--44 42 Stade K, Rinke-Appel J, Brimacombe R (1989) Nucleic Acids Res 17, 9889-9908 43 Oison HM, Lasater LS, Cann PA, Glitz DG (1988) J Bioi Chem 263, 15196-15204 44 Lake JA (1979) In: Transfer RNA: Structure, Properties and Recognition (Schimmel PR, $611 D, Abelson JN, eds) Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 393-411 45 Bollen A, Kahan L, Heimark RL, Cozzone A, Traut RR, Hershey JWB (1975) J Biol Chem 250, 4310--4314 46 Heimark RL, Kahan L, Johnston K, Hershey JWB, Traut RR (1976) J Mol Biol 105,219-230
A model of the tRNA-ribosome complex 47
Olson HM, Grant PG, Cooperman BS, Glitz DG (1982) J
Biol Chem 257, 2649-2656 48 Spirin AS (1983) FEBS Lett 156, 217-221
49
Ofengand J, Lin FL, Hsu L, Boublik M (1981) In:
55 56
Molecular Approaches to Gene and Protein Structure (Siddiqui, MAQ et al, eds) Academic Press, Inc, NY,
57
1-31 50 Gold L (1988) Annu Rev Biochem 57, 199-233 51 Moazed D, Noller I-IF (1986) Cell 47, 985-994 52 Noller HF, Moazed D, Stem S, Powers T, Allen PN, Robertson JM, Weiser B, Triman K (1990) In: The Ribosome: Structure, Function and Evolution (Hill WE, Dahlberg A, Garrett RA, Moore PB, Sehlessinger D, Warner JR, eds) Am Soe Micmbiol Washington, DC, 73-92 53 Wagenkneeht T, Carazo JM, Radermaeher M, Frank J (1989) Biophys J 55,455-464 54 Abdurashidova GG, Tsvetkova EA, Budowky E1 (1990) FEBS Lett 269, 398--401
58 59 60 61 62 63 64
969
Hoppe W, Oettl H, Tietz HR (1986) J Mol Biol 192, 291-322 Girshovich AS, Bochkareva ES, Vasiliev VD (1986) FEBS Lett 197,. 192-198 Kao TH, Miller DL, Abo M, Ofengand J (1983) J Mol Biol 166, 383--405 Podkowinski J, Gomicki P (1989) Nucleic Acids Res 17, 8767-8782 Abdurashidova GG, Tsvetkova EA, Budowsky EI (1989) FEBS Lett 243, 299-302 Lin FL, Kahan L, Ofengand J (1984) J Moi Biol 172, 77-86 Moazed D, Noller HF (1989) Cell 57, 585-597 Langer J, Lake JA (1986) J Mol Biol 187, 617621 Lucas-Lenard J, Tao P, Haenni A (1969) CoM Spring Harbor Syrup Quant Bio134, 455--462 Shorey RL, Ravel JM~ Shive W (1971) Arch Biochem Biophys 146, 110--117
