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Structure-function correlations (and discrepancies) in the 16S ribosomal RNA from Escherichia coli
Biochimie (1992) 74. 319-326 © Soci6t6 fran~aisedc bi~chim~eet biologic mol~ulaire / Elsevier, Pads
319
Structure-function correlations (and discrepancies) in the 16S ribosomal RNA from Escherichia coli R Brimacombe Max-Planck-lnstitutfiir Molekulare Genetik, Abteilung Wittmann,Ihnestrasse 73, lO00 Berlin 33, Germany (Received 26 August 1991; accepted 15 November 1991)
Summary - - The published model for the three-dimensional arrangement of E coli 16S RNA is re-examined in the light of new
experimental information, in particular cross-linking data with functional iigands and cross-links between the 16S and 23S RNA molecules. A growing body of evidence suggests that helix 18 (residues 500-545), helix 34 (residues 1046-1067/!i89-1211) and helix 44 (residues 1409-1491) are incorrectly located in the model. It now appears that the functional sites in helices 18 and 34 may be close to the decoding site of the 30S subunit, rather than being on the opposite side of the 'head' of the subunit, as hitherto supposed. Helix 44 is now clearly located at the interface between the 30S and 50S subunits. Furthermore, almost all of the modified bases in both 16S and 23S RNA appear to form a tight cluster near to the upper end of this helix, surroundingthe decoding site. ribosomal RNA / three-dimensional models / functional sites / electron microscopy / modified bases
Introduction
A few years ago a model was published from this laboratory [1, 21 for the three-dimensional organization of the 16S ribosomal RNA from E coli. The model was based on the topographical information available at the time, including RNA-protein and intra-RNA cross-linking data (summarized in [1 ]), the map of the mass centres of the 30S ribosomal proteins as determined by neutron scattering [3], and several immunoelectron microscopic localizations of individual bases of tile 16S RNA on the surface of the 30S subunit (summarized in [4]). These data were combined with the phylogenetically established secondary structure of the 16S RNA molecule [5, 61 to derive a plausible three-dimensional model. A similar - but by no means identical - model was published a little later by Stem et al [71, which incorporated in addition a substantial set of RNA-protein foot-printing data. Both models [ 1, 7] showed the property that whereas the less highly-conserved regions of the 16S RNA secondary structure were located at the upper and lower extremities of the 30S subunit, the more highly-conserved regions were concentrated into a 'belt' around the centre of the subunit. Sites on the ribosomal RNA that had been implicated in one way or another in the ribosomal function (eg sites of binding of tRNA [8] or antibiotics [9]) were also concentrated into this 'central belt' area, and, moreover, these functional
sites appeared to be divided into two groups, the larger group centred in or near to the 'cleft' between the 'platform' and the 'head' of the 30S subunit, and the other (smaller) group on the opposite side of the 'head'; our 16S model [1], showing this bimodal distribution of the functional sites [10] is shown in figure 1. The separation of the functional sites into two groups in the models was to a large extent a consequence of the immunoelectron microscopic localizations of the methylated G-residue at position 527 in the 16S RNA [16] and of the C-residue at position 1400 [4], which can be cross-linked efficiently to the anticodon loop of P-site bound tRNA [14]; these two positions are both centres of functional importance, but they appeared to be on opposite sides of the 30S subunit [4] (fig 1). It was concluded [7, 10] that the smaller group of functional sites near the position of mG-527 must represent some kind of allosteric phenomenon, whereas the larger 'C-1400' group in the cleft constitutes the actual decoding site. However, more recent data from a number of sources have begun to suggest a direct rather than an allosteric involvement of the functional sites near to mG-527 (fig 1), which in turn implies that there must be serious errors in the three-dimensional 16S RNA models [1, 7]. The purpose of this paper is to describe and discuss these new data, and to indicate those parts of the 16S model that are specifically in need of revision. Three regions of the RNA are concerned,
320 namely helix 18 (residues 500-545), helix 34 (residues 1046-1067/1189-1211) and helix 44 (residues 1409-1491). It is not clear to what extent a relocation of these helices will affect other parts of the structure, and it will therefore take some time before it is possible to present a fully revised version of the model. The electron microscopic evidence
It is appropriate to begin by summarizing the status of the electron microscopic evidence concerning the 527 and 1400 regions of the 16S RNA, because this evidence in itself is by no means unequivocal. Sketches of the appropriate electron microscopic models from the various research groups involved are illustrated in figure 2. The most extreme separation of the two sites
(positions 527 and 1400) is represented by the model of Boublik's group [4], in which the mG-527 is located on the small lobe opposite to the cleft (fig 2A). The 16S RNA could readily be folded in such a way as to meet this criterion [1], and the 'figure 2A-type' location is reflected in the model RNA arrangement of figure 1. (Position 527 and neighbouring nncleotides are shown lying 'outside' the main perimeter of the model in figure 1, because they lie in the singlestranded loop at the end of helix 18, this loop-end is now known to be folded back into the helix in a pseudo-knot interaction ([22], and see figure 3, below).) The RNA model of Stem et al [7] also indicates a wide separation, although not quite so extreme as ours, of the 527 and 1400 positions (fig 22 of [7]). In the electron microscopic model of the St~iffler group [17], the mG-527 is located at the base of the
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Fig 1. The three-dimensional model of 16S RNA, including functional sites, taken from [I0]. The cylinders represent the helical regions of the RNA, numbered as in figure 3. The functional sites are denoted by the small black polygons together with the appropriate position in the 16S sequence, t denotes foot-print sites for tRNA in the absence of a poly(U) template [8], subsequently shown to be P-site positions [ 11]; tu correspondingly denotes sites in the presence of poly(U) subsequently shown to be A-site positions. Str, Spc, Neo etc denote foot-print sites of antibiotics [9], and m indicates a modified nucleotide in the 16S RNA [ 12, 13]. tRNA is the cross-link site to the anticodon loop at position C-1400 [ 14], and poly(A) is the site of cross-linking to a poly(A) messenger analogue at positions 1394-1399 ([10] cf [15]). A. View of the model from the interface side. B. View from the solvent side.
321 head of the subunit rather than on the side lobe (fig 2B), but is still well separated from the presumptive C1400 position at the base of the cleft (the latter position is not in fact included in the model of [17]). In contrast, however, Shatsky and Vasiliev [18] interpret the two sites as being virtually coincident (fig 2C); in this model there is no cleft, but rather a 'ledge' around the base of the head of the subunit. Figures 2D--F show the locations of related sites, interpreted in terms of the 30S subunit model from Lake's group [23]. Figure 2D gives the original location of mG-527 from Glitz's group [ 16], which - as in the St6ffler model of figure 2B - is at the base of the head on the opposite side to the cleft. Figure 2E summarizes the results of Iocalizations by Lake's group [19, 20] of three RNA positions, namely C-518, G-1392 and A-1492, using the techLaique of DNA hybridization electron microscopy. C-518 is in the same hairpin loop-end as mG-527 (see figure 3, below), and by virtue of the pseudoknot just mentioned - is very close to the latter position. G-1392 is similarly close to C-1400, and this area is furthermore connected to the 1500-region (see below) both by phylogenetically established tertiary interactions [22] and by intraRNA cross-links [47]. In agreement with the 1400-1500 neighbourhood, figure 2E indicates that positions 1392 and 1492 map at indistinguishable sites [20] which correspond to the location of C-1400 near to the base of the cleft (fig 2A). However, it should be noted that in this case (fig 2E) the sites are indicated as being on the solvent side rather than the interface side of the 30S subunit. Furthermore, in one set of images (fig 5D in [19]) the 1392 probe was observed on the opposite side of the head, effectively in the 'mG-527 position' (as in fig 2D). The location of C-518 in figure 2E is similar to that of mG-527 in figure 2D. although again the site mapped extends towards the solvent side of the subunit rather than the interface side, and indeed overlaps with the G-1392/A-1492 sites. Despite this interchangeability and overlap of the two sites, Oakes et al [20] interpreted the arrangement of the 16S RNA so as to give the maximal separation of the 1392 and 518 regions that was compatible with their observations (as do the models of [1, 71 as already noted). Figure 2F gives the position observed by Montesano and Glitz [21] of N6-monomethyladenosine in wheat-germ ribosomes. This nucleotide clearly maps at the 'mG-527' position (cf fig 2D), but - by comparison with the sequence of Xenopus laevis 18S RNA [24] - is at a position corresponding to A-1500 in the E coli 16S RNA, and should therefore map on the cleft side (as does the A-1492 in fig 2E). In the same publication [21], the authors also mapped the m7G residue from wheat germ at the same location (cf fig 2D), although in eukaryotes [25] this nucleotide is at a
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Fig 2. Sketches of models of the E coli 305 ribosomal subunit, derived from immuno-electron microscopic studies, showing the locations deduced for mG-527, C-1400 and related positions. A, B, C. The models of [4, 17, 18] respectively. I). The location of mG-527, according to [16]. E. The locations of G-1392, A-1492 and C-518, determined by DNA hybridization electron microscopy [19, 20]. F. The location of mono-methyl adenosine on the eukaryotic 40S subunit [21]; this nucleotide is at a position corresponding to A-1500 in E coll. The views in A, B, C, D and F are from the interface side of the subunit, that in E from the solvent side.
position corresponding to G-1338 of the E coli sequence (see fig 3, below). The latter nucleotide has been cross-linked to protein S13 [26], and lies at a position in the 16S RNA (fig 3) which also favours a location on the cleft side of the head of the 30S subunit (cfthe model of fig 1). From these conflicting results it can only be concluded that, although the 527 and 1400 regions do indeed both appear to lie at the base of the head of the 30S subunit, the electron microscopic techniques are not capable of distinguishing between the two positions in this particular instance. In this ~.ontext it is important to remember that the head of the subunit is only joined to the body by a single helix of the 16S RNA (helix 28, fig 3, and see [1, 7]), and that the head can be severed from the body and isolated as a ribonucleoprotein fragment under very mild conditions of ribonuclease digestion [27]. The 'neck' of the 30S subunit may thus be much narrower than the electron microscopic models suggest, which would in turn imply that the two regions (527 and 1400) do in fact
322 Biochemical evidence 527 and 1400 regions
lie close together in the three-dimensional structure o f the 16S R N A . This c o m m o n location will for convenience be referred to simply as the decoding site; whether this site is indeed in the cleft, or further around the subunit (as in fig 2C), remains unclear.
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Fig 3, The secondary structure of E coli 16S RNA [28], showing topographical information relevant to this paper. Tertiary interactions [22] are indicated by fine lines connecting the interacting bases (eg positions 1399-1504). Intra-RNA cross-links are denoted by heavier lines, numbered with Roman numerals. Filled squares represent the foot-print sites for protein S 12 [29], open triangles the foot-print sites for streptomycin [9], and open circles sites of mutation causing resistance to streptomycin [30-33]. Filled circles arc foot-print sites for P-site bound tRNA [1 l], filled triangles the corresponding sites for A-site bound tRNA. SmX denotes sites of cross-linking to streptomycin [34], and SpcR and SpcF are sites of resistance [35] and foot-printing [9] to spectinomycin. ~spectively. tRNA-X is the site of cross-linking to the anticodon loop of tRNA [14]. mRNA (_+ n) are positions of site-directed cross-linking to mRNA analogues [15, 36, 37]; the number in brackets indicates the position of the cross-link within the mRNA, upstream (-) or downstrear-" (+) from the 5'-position of the P-site codon (The sites at positions 1392-1398 and at the extreme 3'-terminus of the 16S RNA were observed at various + and - positions, respectively [15, 36].) m denotes a modified nucleotide in the 16S RNA [12, 13].
323 16S RNA, namely data relating to the antibiotic streptomycin, foot-printing results for protein S12, footprinting results for tRNA at the ribosomal A- or Psite, and cross-linking data with mRNA analogues. A fifth line of evidence involving the clustering of modified bases in the 16S and 23S RNA is considered in a separate section below. Figure 3 shows the secondary structure of the 16S RNA [281, and includes the data sets relevant to this discussion; for most of the sites concerned, the corresponding positions in the three-dimensional model of the RNA [ll can be seen in figure 1. Mutations causing resistance to streptomycin have been found at several sites in the 16S RNA, These include positions 507, 523 and 525 in helix 18 [31, 33], and positions 13 and 912-915 in the neighbourhood of helix 2 [30, 32]. It should be noted that position 507 is directly connected to 524 via the pseudoknot interaction noted above ([22, 331 and see fig 3). Foot-printing studies with the antibiotic 191 showed effects at positions 911-915, coincident with the second group of resistance sites, and also 'Class Ill effects' [9] at positions 1413 and 1487 in helix 44 (see below). Streptomycin has also been cross-linked to the 16S RNA, and two cross-link sites were localized to residues 892-917 (covering helices 2 and 27) and to residues 1392-1415 [34]. A further piece of evidence is the observation made many years ago [38] that streptomycin inhibits the cleavage by colicin E3 of the 16S RNA at position 1493; as already discussed above, this position is in close proximity to the 14~qregion. Thus the streptomycin data form a link between helix 18, the region of helices 2 and 27, and the 1400-region. The relationship between streptomycin and ribosomal protein Si2 has recently been discussed in detail by Powers and Noller [33]. All that needs to be mentioned here is the fact that this protein gives a foot-print [29] on 16S RNA in helix 18 and in the region of helices 19 and 27 (fig 3), and also shows effects at nucleotides 1392-1396. Thus, just as with the antibiotic data, these results establish a connection between the 527 and 1400 regions. The tRNA foot-printing data present a similar picture. Most of the foot-print sites [ 11 ] are concentrated in the cleft region of the RNA model (fig l), and in some cases there is direct evidence for the juxtaposition of the individual foot-print sites, such as those at positions 693 and 794-795 which are connected by the intra-RNA cross-link I (fig 3), or those in the region of helix 44 which are connected by phylogenetically established tertiary interactions and by the intra-RNA cross-link III (fig 3, and see below). Similar or even more extensive inter-connections between the corresponding tRNA foot-print sites in 23S RNA [39] have also been noted [40, 411. Again,
however, the data provide a link between the 527 and 1400 regions, with P-site bound tRNA showing footprints at positions 532 and 1399-1401 11 ~], and A-site bound tRNA showing foot-prints at positions 529531, 1408 and 1492-1493 [11]. Furthermore, the antibiotic neomycin mimics the A-site tRNA, showing foot-print effects at positions 525, 1408 and 1494 [9]. Although it is difficult to formally exclude the possibility that the effects observed in the 530 loop are allosteric, it seems highly unlikely that such different ligands as tRNA, protein S12, and two antibit~tics should all be able to cause allosteric effects at similar but not identical sites (a common conformational change induced by all the ligands would rather be expected to show effects at identical positions). The fourth line of evidence comes from sitedirected cross-linking experiments with mRNA [15], where an mRNA analogue carrying a thio-uridine residue at position +11 (relative to the 5'-base of the P-site codon) showed concomitant cross-linking to positions 532 and to positions 1392-1398, (the latter positions also having been observed in older crosslinking studies with a simple poly(A) messenger analogue [10], see fig 1). Here, it could be argued that two distinct binding sites for the mRNA were simultaneously involved, but, taken together with the data described above, this result provides additional circumstantial evidence for a juxtaposition of the 527 and 1400 regions. At all events, it seems dear that the evidence in favour of this juxtaposition now outweighs the somewhat self-contradictory electron microscopic evidence for a physical separation of the two areas, as discussed in the previous section.
The location of helix 34 If, as just suggested, the loop-end of helix 18 is to be relocated at the decoding site of the RNA model, then this would only leave the functional sites that have been observed in helix 34 on the 'wrong' side of the 30S subunit (fig 1). Helix 34 (fig 3) contains a site of resistance to the antibiotic spectinomycin [35], as well as foot-print sites to the latter [9] and a modified nucleotide (see below). However, there is now direct evidence that helix 34 lies close to the decoding site, again from site-directed cross-linking studies with mRNA analogues [37]. In this case, a thio-uridine residue located at position +6 relative to the 5'-base of the P-site codon in the mRNA was found to be involved in a tRNA-dependent cross-link to position ca 1055 of the 16S RNA in the 30S initiation complex. The position of the cross-link has since been precisely localized to position 1052, and has also been observed in the 70S initiation complex (Olga Dontsova, personal communication). Position +6 corre-
324 sponds to the 3'-base of the A-site codon, and this result thus places position 1052 in helix 34 directly in the decoding site of the 30S subunit. It should also be noted that this cross-link to helix 34 is only five bases in the mRNA away from that at the +11 position described in the previous section, which was crosslinked to position 532 of the 16S RNA. Helix 34 has been implicated in the termination event of protein synthesis, by the finding that a deletion at position 1054 causes suppression of the UGA termination signal 1421, and a model was proposed involving direct base-pairing between the UGA triplet and two tandem complementary UCA sequences in the opposing strand of helix 34. It now appears that in fact this deletion causes read-through of all three termination triplets, which casts doubt on the base-pairing model (Catherine Prescott, personal communication), but at the same time indicates a more universal involvement of helix 34 in termination. Since a base change in yeast mitochondria at a site corresponding to position 517 in E coli 16S RNA has been shown to cause supression of an ochre mutation [431, this provides a further circumstantial link between the 530 region and helix 34, which, as just described, must now be relocated to the decoding site of the 30S subunit.
The environment of helix 44, and clustering of modified nucleotides The location of helix 44, which is adjacent to the 1400 region of the 16S RNA (fig 3), is clearly relevant to any discussion of the topography of the ribosomal decoding site. Since publication of the RNA model [1 ], a number of observations have been made conceming the environment of this helix, some of which have already been mentioned in the preceding sections. First, the phylogenetically conserved tertiary interactions that have been reported between bases 1399 and 1495, 1401 and 1501, and 1405 and 1504 I221, impose important constraints on the singlestranded regions of RNA on either side of helix 44, and further confirmation of these constraints is provided by the intra-RNA cross-link III ([471; fig 3). Another intra-RNA cross-link has also been observed in this area [47] between positions 1393-1401 and 1531-1542 (cross-link II, fig 3), which effectively joins the extreme 3'-terminus of the 16S RNA with the 1400 region. This cross-link most probably reflects a flexibility of the T-terminus, which we have previously commented on [ 1], and raises a note of caution as to the placement of the T-terminus by electron microscopy [eg 44] as well as to the interpretation of its involvement in cross-links to mRNA analogues at positions upstream of the coding triplet [36].
Most important, however, is the very recent finding that bases 1408-1411 (at the left-hand end of helix 44, fig 3) are involved in a direct cross-link to 23S RNA at the 30S-50S subunit interface [48]. This indicates that helix 44 is incorrectly placedin the model (fig 1), and that at least its upper end should be relocated on the interface side of the 30S subunits, as opposed to the solvent side. Such a placement would bring the upper end of helix 44 (bases 1409/1491) close to the loop-end of helix 45 (see fig 1; the loop-end of this latter helix points downward in the model), and in fact this loop-end (an oligonucleotide covering positions 1518-1523) was also cross-linked to 23S RNA [48] at a site very close to or identical with that cross-linked to positions 1408-1411 of the 16S. Other recent observations support a location of helix 44 at the interface [48]. This raises the interesting question of the clustering of the modified nucleotides in the 16S and 23S RNA at the functional centres of the two subunits, which now appears to be much more extreme than hitherto supposed [I, 10]. In the 16S RNA, cross-link Ill and the tertiary interactions bring the three modified bases at positions 1402, 1407 and 1498 into close proximity with one another, and the interface cross-links just mentioned bring the further three modified bases at the loop-end of helix 45 (positions 1516, 1518 and 1519) into the same near neighbourhood. The two modified nucleotides at the loop-end of helix 31 (positions 966, 967) also appear to lie in the cleft of the subunit (fig 1), and - if the arguments presented in the previous sections are correct - then the remaining two modified nucleotides at position 527 (in the loop-end of helix 18) and position 1207 (in helix 34) also belonl~ to this cluster. Thus, all of the modified bases in the 16S RNA would then lie in the immediate vicinity of the decoding site. The situation in the 23S RNA is very similar. Nineteen modified nucleotides have now been identified in the E coli 23S RNA molecule (Cooperman B, Mitchell P, in preparation), and no less than eighteen of these are at positions which are either connected by direct intra-RNA cross-links [40, 41] or lie close to tRNA foot-print sites [39]. (The nineteenth, at position 1608 of the 23S RNA sequence, lies in a region which is so far devoid of topographical information, and thus may or may not also belong to the cluster.) Most dramatic, however, is the information provided by the interface cross-links discussed above; the site in the 23S RNA involved in these cross-links covers positions 1911-1920, where no less than three of the modified bases are located. Furthermore, both interface cross-links (to positions 14081411 and positions 1518-1523 of the 16S RNA) were also observed when polysomes were used as the substrate for the cross-linking reaction. Thus, the vast
325
majority, if not all, of the modified nucleotides in 16S and 23S RNA form a tight topographical cluster which is directly related to the binding site of tRNA and forms the active centre of the ribosome. A substantial proportion of the invariant bases in the 16S and 23S RNA also contribute to this cluster [48].
6 7 8 9
Conclusions The published three-dimensional models of the 16S ribosomal RNA [1, 7] have relied heavily on the results of immunoelectron microscopic studies (eg [41) and on data concerning the arrangement of the ribosomal proteins - in particular the neutron scattering map [3] - in order to locate the various RNA helices in the structure. However, because so little is known about the sizes and shapes of the individual proteins, the placement of an element of the RNA structure in the model on the basis of the corresponding position of a particular protein is subject to a large inherent error, and the resolution provided by the electron microscopic approach is similarly low. The arguments presented here suggest that in consequence several helices in the published models are incorrectly located, but some of the evidence for this is still circumstantial, being based on foot-printing type data, rather than on unambiguously direct observations and measurements. A further exploitation of the various cross-linking approaches, in particular site-directed cross-linking of functional ligands such as mRNA [15, 37] or tRNA [45, 46] as well as the precise analysis of intra-RNA cross-links within the 16S and 23S molecules themselves (eg [411), seems to offer the most promising approach for finally resolving these discrepancies and for developing the next generation of three-dimensional models for the ribosomal RNA.
10
11 12 13
14
15
16
17
18 19
References I Brimacombe R, Atmadja J, Stiege W, Schuler D (1988) A
2
3 4
5
detailed model of the three-dimensional structure of E coli 16S ribosomal RNA in situ in the 30S subunit. J Mot Biol 199, 115-136 SchUler D, Brimacombe R (1988) The E coli 30S ribosomal subunit; an optimized three-dimensional fit between the ribosomal proteins and the 16S RNA. EMBO J 7, 1509-!513 Capel MS, Kjeidgaard M, Engelman DM, Moore PB (1988) Positions of $2, S13, S16, S17, S19 and $21 in the 30S ribosomal subunit of E coli. J Mot Bio1200, 65-87 Gomicki P, Nurse K, Hellmann W, Boublik M, Ofengand J (1984) High resolution localization of the tRNA anticodon interaction site on the E coli 30S ribosomal subunit. J Biol Chem 259, 10493-10498 Noller HF (1984) Structure of ribosomal RNA. Annu Rev Biochem 53, 119-162
20 21
22 23
Brimacombe R, Stiege W (1985) Structure and function of ribosomal RNA. Biochem J 229, 1-17 Stem S, Weiser B, Noller HF (1988) Model for the threedimensional folding of 16S ribosomal RNA. J Mol Blot 204, 447--481 Moazed D, Noller HF (1986) Transfer RNA shields specific nucleotides in 16S ribosomal RNA from attack by chemical probes. Cell 47, 985-994 Moazed D, Noller HF (1987) Interaction of antibiotics with functional sites in 16S ribosomal RNA. Nature 327, 389-394 Stiege W, Stade X, Schuler D, Brimacombe R (1988) Covalent cross-linking of poly(A) to E coil ribosomes, and localization of the cross-link site within the 16S RNA. Nucleic Acids Res 16, 2369-2388 Moazed D, Noller HF (1990) Binding of tRNA to the ribosomal A and P sites protects two distinct sets of nucleotides in 16S RNA. J Mot Biol 21 I, 135-145 Carbon P, Ehresmann C, Ehresnlann B, Ebel JP (1979) The complete nucleotide sequence of the ribosomal 16S RNA from E coil Fur J Biochem 100, 399--410 Van Knippenberg PH, Van Kimmenade JMA, Heus JA (1984) Phylogeny of the conserved 3' terminal structure of the RNA of small ribosomal subunits. Nucleic Acids Res 12, 2595-2604 Prince JB, Taylor BH, Thurlow DL, Ofengand J, Zimmermann RA (1982) Covalent cross-linking of tRNA TM to 16S RNA at the ribosomal P-site; identification of crosslinked residues. Proc Natl Acad Sci USA 79, 5450-5454 Rinke-Appel J, Jiinke N, Stade K, Brimacombe R (1991) The path of ,nRNA through the E coli ribosome; sitedirected cross-linking of mRNA analogues carrying a photoreactive label at various points 3' to the decoding site. EMBO J 10, 2195-2202 Trempe MR, Oghi K, Glitz DG (1982) Localization of 7-methylguanosine in the small subunits of E coli and chloroplast ribosomes by immuno-electron microscopy. J Biol Chem 257, 9822-9829 Sttiffler G, Sttiffler-Meilicke M (1986) Immuno-electron microscopy on E coli ribosomes. In: Strut'ture. Fum'tioo arm Genetit's of Ribosomes (Hardesty B, Kramer G, eds) Springer-Verlag, New York, 28--46 Shatsky IN, Bakin AV, Bogdanov AA, Vasiliev VD (1991) How does the mRNA pass through the ribosome? Biochimie, 73.937-945 Oakes MI, Clark MW, Henderson E, Lake JA (1986) DNA hybridization electron microscopy; ribosomal RNA nucleotides 1392-1407 are exposed in the cleft of the small subunit. Proc Natl Acad Sci USA 83,275-279 Oakes MI, Kahan L, Lake JA (1990) DNA hybridization electron microscopy; tertiary structure of 16S rRNA. J Mot Biol 211,907-918 Montesano L, Glitz DG (1988) Wheat germ cytoplasmic ribosomes; localization of 7-methylguanosine and 6-methyladenosine by electron microscopy of immune complexes. J Biol Chem 263, 4939---4944 Haselman T, Camp DG, Fox GE (1989) Phylogenetic evidence for tertiary interactions in 16S-like ribosomal RNA. Nucleic Acids Res 17, 2215-2221 Oakes M, Henderson E, Scheinman A, Clark M, Lake JA (1986) Ribosome structure, function and evolution; mapping ribosomal RNA, proteins and functional sites in three dimensions. In: Structure, Function and Genetics of Ribosomes (Hardesty B, Kramer G, eds) Springer-Verlag, New York, 47--67
326 24 25
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33 34 35 36
37
Maden BEH (1986) Identification of the locations of the methyl groups in 18S RNA from X laevis and man. J Mol Bioi 189, 681-699 Zueva VS, Mankin AS, Bogdanov AA, Thurlow DL, Zimmermann RA (1985) Occurrence and location of 7methylguanine residues in small-subunit ribosomal RNAs from eubacteria, archaebacteria and eukaryotes. FEBS Lett 188, 233-238 Osswald M, Greuer B, Brimacombe R, Strffler G, Biiumert H, Fasold H (1987) RNA-protein cross-linking in E coli ribosomal subunits; determination of sites on 16S RNA that are cross-linked to proteins $3, $4, $5, $7, $8, $9, SI1, S13, SI9 and $21 by treatment with methyl-pazidophenyl acetimidate. Nucleic" Acids" Res 15, 32213240 Yuki A, Brimacombe R (1975) Nucleotide sequences of E coli 16S RNA associated with ribosomal proteins $7, $9, SI0. S14 and SI9. E u r J Biochem 56, 23-34 Brimacombe R (1991) RNA-protein interactions in the E coli ribosome. Biochimie, 73,927-936 Stem S. Powers T, Changchien LM. Noller HF (1988) Interaction of ribosomal proteins $5, $6, Sll, S12, S18 and $21 with 16S rRNA. J Mol Biol 201,683-695 Montandon PE, Nicolas P, Schiirmann P, Stutz E (1985) Streptomycin resistance of E gracilis chloroplasts; identification of a point mutation in the 16S rRNA gene at an invariant position. Nucleic Acids Res 13, 4299-4310 Melan~on P, Lemieux C, Brakier-Gingras L (1988) A mutation in the 530 loop of E coil 16S ribosomal RNA causes resistance to streptomycin. Nucleic Acids Res 16, 9631-9639 Harris Eli, Burkhart BD, Gillham NW, Boynton JE (1989) Antibiotic resistance mutations in the chloroplast 16S and 23S rRNA genes of C reinhardtii; correlation of genetic and physical maps of the chloroplast genome. Genetics 123, 281-292 Powers T, Noller HF (1991) A functional pseudoknot in 16S ribosomal RNA. EMBO J IO, 2203-2214 Gravel M, Melanqon P, Brakier-Gingras L (1987) Crosslinking of streptomycin to the 16S ribosomal RNA of E coli. Biochemistry 26, 6227-6232 Sigmund CD, Ettayebi M, Morgan EA (1984) Antibiotic resistance mutations in 16S and 23S ribosomal RNA genes of E coll. Nucleic Acids Res 12, 4653-4663 Stade K, Rinke-Appel J, Brimacombe R (1989) Sitedirected cross-linking of mRNA analogues to the E coli ribosome; identification of 30S ribosomal components that can be cross-linked to the mRNA at points 5' with respect to the decoding site. Nucleic Acids Res 17, 9889-9908 Dontsova O, Kopylov A, Brimacombe R (1991) The location of mRMA in the 30S initiation complex; site-
38
39 40
41
42 43
44
45
46
47
48
directed cross-linking of mRNA analogues carrying several photo-reactive labels simultaneously on either side of the AUG start codon. E M B O J 10, 2613-2620 Dahlberg AE, Lund E, Kjeldgaard NO, Bowman CM, Nomura M (1973) Colicin E3 induced cleavage of 16S ribosomal RNA; blocking effects of certain antibiotics. Biochemistry 12, 948-950 Moazed D, Noller HF (1989) Interaction of tRNA with 23S rRNA in the ribosomal A, P and E sites. Cell 57, 585-597 Mitchell P, Osswald M, Schiller D, Brimacombe R (1990) Selective isolation and detailed analysis of intra-RNA cross-links induced in the large ribosomal subunit of E coli; a model for the tertiary structure of the tRNA binding domain in 23S RNA. Nucleic Acids Res 18, 4325-4333 Drring T, Greuer B, Brimacombe R (1991) The threedimensional folding of ribosomal RNA; localization of a series of intra-RNA cross-links in 23S RNA induced by treatment of E coil 50S ribosomal subunits with bis-(2chloroethyl) methylamine. Nucleic Acids Res 19, 35173524 Murgola El, Hijazi KA, Grringer U, Dahlberg AE (1988) Mutant 16S ribosomal RNA; a codon-specific translational suppressor. Proc Natl Acad Sci USA 85, 4162--4165 Sben Z, Fox TD (1989) Substitution of an invariant nucleotide at the base of the highly conserved 530-1oop of 15S rRNA causes suppression of yeast mitochondrial ochre mutations. Nucleic Acids Res 17, 4535-4539 LUhrmann R, StOffler-Meilicke M, Strffier G (1981) Localization of the 3'-end of 16S rRNA in E coli 30S ribosomal subunits by immuno-electron microscopy. Mol Gen Genet 182, 369-376 WowerJ, Hixson SS, Zimmermann RA (1989) Labeling the peptidyl transferase center of the E coil ribosome with photoreactive tRNAphe derivatives containing azidoadenosine at the 3' end of the acceptor ann; a new model of the tRNAribosome complex. Proc Natl Acad Sci USA 86, 5232-5236 Podkowinski J, Gomicki P (1989) Ribosomal proteins $7 and LI are located close to the decoding site of E coli ribosomes; affinity labelling studies with modified tRNAs carrying photoreactive probes attached adjacent to the T-end of the anticodon. Nucleic Acids Res 17, 87678782 Drring T, Greuer B, Brimacombe R (1992) The topography of the 3'-terminal region of E coil 16S ribosomal RNA; an intra-RNA cross-linking study. Nucleic Acids Res, in press Mitchell P, Osswald M, Brimacombe R (1992) Identification of intermolecular RNA cross-links at the subunit interface of E coli ribosome. Biochemistry, in press
319
Structure-function correlations (and discrepancies) in the 16S ribosomal RNA from Escherichia coli R Brimacombe Max-Planck-lnstitutfiir Molekulare Genetik, Abteilung Wittmann,Ihnestrasse 73, lO00 Berlin 33, Germany (Received 26 August 1991; accepted 15 November 1991)
Summary - - The published model for the three-dimensional arrangement of E coli 16S RNA is re-examined in the light of new
experimental information, in particular cross-linking data with functional iigands and cross-links between the 16S and 23S RNA molecules. A growing body of evidence suggests that helix 18 (residues 500-545), helix 34 (residues 1046-1067/!i89-1211) and helix 44 (residues 1409-1491) are incorrectly located in the model. It now appears that the functional sites in helices 18 and 34 may be close to the decoding site of the 30S subunit, rather than being on the opposite side of the 'head' of the subunit, as hitherto supposed. Helix 44 is now clearly located at the interface between the 30S and 50S subunits. Furthermore, almost all of the modified bases in both 16S and 23S RNA appear to form a tight cluster near to the upper end of this helix, surroundingthe decoding site. ribosomal RNA / three-dimensional models / functional sites / electron microscopy / modified bases
Introduction
A few years ago a model was published from this laboratory [1, 21 for the three-dimensional organization of the 16S ribosomal RNA from E coli. The model was based on the topographical information available at the time, including RNA-protein and intra-RNA cross-linking data (summarized in [1 ]), the map of the mass centres of the 30S ribosomal proteins as determined by neutron scattering [3], and several immunoelectron microscopic localizations of individual bases of tile 16S RNA on the surface of the 30S subunit (summarized in [4]). These data were combined with the phylogenetically established secondary structure of the 16S RNA molecule [5, 61 to derive a plausible three-dimensional model. A similar - but by no means identical - model was published a little later by Stem et al [71, which incorporated in addition a substantial set of RNA-protein foot-printing data. Both models [ 1, 7] showed the property that whereas the less highly-conserved regions of the 16S RNA secondary structure were located at the upper and lower extremities of the 30S subunit, the more highly-conserved regions were concentrated into a 'belt' around the centre of the subunit. Sites on the ribosomal RNA that had been implicated in one way or another in the ribosomal function (eg sites of binding of tRNA [8] or antibiotics [9]) were also concentrated into this 'central belt' area, and, moreover, these functional
sites appeared to be divided into two groups, the larger group centred in or near to the 'cleft' between the 'platform' and the 'head' of the 30S subunit, and the other (smaller) group on the opposite side of the 'head'; our 16S model [1], showing this bimodal distribution of the functional sites [10] is shown in figure 1. The separation of the functional sites into two groups in the models was to a large extent a consequence of the immunoelectron microscopic localizations of the methylated G-residue at position 527 in the 16S RNA [16] and of the C-residue at position 1400 [4], which can be cross-linked efficiently to the anticodon loop of P-site bound tRNA [14]; these two positions are both centres of functional importance, but they appeared to be on opposite sides of the 30S subunit [4] (fig 1). It was concluded [7, 10] that the smaller group of functional sites near the position of mG-527 must represent some kind of allosteric phenomenon, whereas the larger 'C-1400' group in the cleft constitutes the actual decoding site. However, more recent data from a number of sources have begun to suggest a direct rather than an allosteric involvement of the functional sites near to mG-527 (fig 1), which in turn implies that there must be serious errors in the three-dimensional 16S RNA models [1, 7]. The purpose of this paper is to describe and discuss these new data, and to indicate those parts of the 16S model that are specifically in need of revision. Three regions of the RNA are concerned,
320 namely helix 18 (residues 500-545), helix 34 (residues 1046-1067/1189-1211) and helix 44 (residues 1409-1491). It is not clear to what extent a relocation of these helices will affect other parts of the structure, and it will therefore take some time before it is possible to present a fully revised version of the model. The electron microscopic evidence
It is appropriate to begin by summarizing the status of the electron microscopic evidence concerning the 527 and 1400 regions of the 16S RNA, because this evidence in itself is by no means unequivocal. Sketches of the appropriate electron microscopic models from the various research groups involved are illustrated in figure 2. The most extreme separation of the two sites
(positions 527 and 1400) is represented by the model of Boublik's group [4], in which the mG-527 is located on the small lobe opposite to the cleft (fig 2A). The 16S RNA could readily be folded in such a way as to meet this criterion [1], and the 'figure 2A-type' location is reflected in the model RNA arrangement of figure 1. (Position 527 and neighbouring nncleotides are shown lying 'outside' the main perimeter of the model in figure 1, because they lie in the singlestranded loop at the end of helix 18, this loop-end is now known to be folded back into the helix in a pseudo-knot interaction ([22], and see figure 3, below).) The RNA model of Stem et al [7] also indicates a wide separation, although not quite so extreme as ours, of the 527 and 1400 positions (fig 22 of [7]). In the electron microscopic model of the St~iffler group [17], the mG-527 is located at the base of the
t,::e
A
Fig 1. The three-dimensional model of 16S RNA, including functional sites, taken from [I0]. The cylinders represent the helical regions of the RNA, numbered as in figure 3. The functional sites are denoted by the small black polygons together with the appropriate position in the 16S sequence, t denotes foot-print sites for tRNA in the absence of a poly(U) template [8], subsequently shown to be P-site positions [ 11]; tu correspondingly denotes sites in the presence of poly(U) subsequently shown to be A-site positions. Str, Spc, Neo etc denote foot-print sites of antibiotics [9], and m indicates a modified nucleotide in the 16S RNA [ 12, 13]. tRNA is the cross-link site to the anticodon loop at position C-1400 [ 14], and poly(A) is the site of cross-linking to a poly(A) messenger analogue at positions 1394-1399 ([10] cf [15]). A. View of the model from the interface side. B. View from the solvent side.
321 head of the subunit rather than on the side lobe (fig 2B), but is still well separated from the presumptive C1400 position at the base of the cleft (the latter position is not in fact included in the model of [17]). In contrast, however, Shatsky and Vasiliev [18] interpret the two sites as being virtually coincident (fig 2C); in this model there is no cleft, but rather a 'ledge' around the base of the head of the subunit. Figures 2D--F show the locations of related sites, interpreted in terms of the 30S subunit model from Lake's group [23]. Figure 2D gives the original location of mG-527 from Glitz's group [ 16], which - as in the St6ffler model of figure 2B - is at the base of the head on the opposite side to the cleft. Figure 2E summarizes the results of Iocalizations by Lake's group [19, 20] of three RNA positions, namely C-518, G-1392 and A-1492, using the techLaique of DNA hybridization electron microscopy. C-518 is in the same hairpin loop-end as mG-527 (see figure 3, below), and by virtue of the pseudoknot just mentioned - is very close to the latter position. G-1392 is similarly close to C-1400, and this area is furthermore connected to the 1500-region (see below) both by phylogenetically established tertiary interactions [22] and by intraRNA cross-links [47]. In agreement with the 1400-1500 neighbourhood, figure 2E indicates that positions 1392 and 1492 map at indistinguishable sites [20] which correspond to the location of C-1400 near to the base of the cleft (fig 2A). However, it should be noted that in this case (fig 2E) the sites are indicated as being on the solvent side rather than the interface side of the 30S subunit. Furthermore, in one set of images (fig 5D in [19]) the 1392 probe was observed on the opposite side of the head, effectively in the 'mG-527 position' (as in fig 2D). The location of C-518 in figure 2E is similar to that of mG-527 in figure 2D. although again the site mapped extends towards the solvent side of the subunit rather than the interface side, and indeed overlaps with the G-1392/A-1492 sites. Despite this interchangeability and overlap of the two sites, Oakes et al [20] interpreted the arrangement of the 16S RNA so as to give the maximal separation of the 1392 and 518 regions that was compatible with their observations (as do the models of [1, 71 as already noted). Figure 2F gives the position observed by Montesano and Glitz [21] of N6-monomethyladenosine in wheat-germ ribosomes. This nucleotide clearly maps at the 'mG-527' position (cf fig 2D), but - by comparison with the sequence of Xenopus laevis 18S RNA [24] - is at a position corresponding to A-1500 in the E coli 16S RNA, and should therefore map on the cleft side (as does the A-1492 in fig 2E). In the same publication [21], the authors also mapped the m7G residue from wheat germ at the same location (cf fig 2D), although in eukaryotes [25] this nucleotide is at a
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Fig 2. Sketches of models of the E coli 305 ribosomal subunit, derived from immuno-electron microscopic studies, showing the locations deduced for mG-527, C-1400 and related positions. A, B, C. The models of [4, 17, 18] respectively. I). The location of mG-527, according to [16]. E. The locations of G-1392, A-1492 and C-518, determined by DNA hybridization electron microscopy [19, 20]. F. The location of mono-methyl adenosine on the eukaryotic 40S subunit [21]; this nucleotide is at a position corresponding to A-1500 in E coll. The views in A, B, C, D and F are from the interface side of the subunit, that in E from the solvent side.
position corresponding to G-1338 of the E coli sequence (see fig 3, below). The latter nucleotide has been cross-linked to protein S13 [26], and lies at a position in the 16S RNA (fig 3) which also favours a location on the cleft side of the head of the 30S subunit (cfthe model of fig 1). From these conflicting results it can only be concluded that, although the 527 and 1400 regions do indeed both appear to lie at the base of the head of the 30S subunit, the electron microscopic techniques are not capable of distinguishing between the two positions in this particular instance. In this ~.ontext it is important to remember that the head of the subunit is only joined to the body by a single helix of the 16S RNA (helix 28, fig 3, and see [1, 7]), and that the head can be severed from the body and isolated as a ribonucleoprotein fragment under very mild conditions of ribonuclease digestion [27]. The 'neck' of the 30S subunit may thus be much narrower than the electron microscopic models suggest, which would in turn imply that the two regions (527 and 1400) do in fact
322 Biochemical evidence 527 and 1400 regions
lie close together in the three-dimensional structure o f the 16S R N A . This c o m m o n location will for convenience be referred to simply as the decoding site; whether this site is indeed in the cleft, or further around the subunit (as in fig 2C), remains unclear.
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Fig 3, The secondary structure of E coli 16S RNA [28], showing topographical information relevant to this paper. Tertiary interactions [22] are indicated by fine lines connecting the interacting bases (eg positions 1399-1504). Intra-RNA cross-links are denoted by heavier lines, numbered with Roman numerals. Filled squares represent the foot-print sites for protein S 12 [29], open triangles the foot-print sites for streptomycin [9], and open circles sites of mutation causing resistance to streptomycin [30-33]. Filled circles arc foot-print sites for P-site bound tRNA [1 l], filled triangles the corresponding sites for A-site bound tRNA. SmX denotes sites of cross-linking to streptomycin [34], and SpcR and SpcF are sites of resistance [35] and foot-printing [9] to spectinomycin. ~spectively. tRNA-X is the site of cross-linking to the anticodon loop of tRNA [14]. mRNA (_+ n) are positions of site-directed cross-linking to mRNA analogues [15, 36, 37]; the number in brackets indicates the position of the cross-link within the mRNA, upstream (-) or downstrear-" (+) from the 5'-position of the P-site codon (The sites at positions 1392-1398 and at the extreme 3'-terminus of the 16S RNA were observed at various + and - positions, respectively [15, 36].) m denotes a modified nucleotide in the 16S RNA [12, 13].
323 16S RNA, namely data relating to the antibiotic streptomycin, foot-printing results for protein S12, footprinting results for tRNA at the ribosomal A- or Psite, and cross-linking data with mRNA analogues. A fifth line of evidence involving the clustering of modified bases in the 16S and 23S RNA is considered in a separate section below. Figure 3 shows the secondary structure of the 16S RNA [281, and includes the data sets relevant to this discussion; for most of the sites concerned, the corresponding positions in the three-dimensional model of the RNA [ll can be seen in figure 1. Mutations causing resistance to streptomycin have been found at several sites in the 16S RNA, These include positions 507, 523 and 525 in helix 18 [31, 33], and positions 13 and 912-915 in the neighbourhood of helix 2 [30, 32]. It should be noted that position 507 is directly connected to 524 via the pseudoknot interaction noted above ([22, 331 and see fig 3). Foot-printing studies with the antibiotic 191 showed effects at positions 911-915, coincident with the second group of resistance sites, and also 'Class Ill effects' [9] at positions 1413 and 1487 in helix 44 (see below). Streptomycin has also been cross-linked to the 16S RNA, and two cross-link sites were localized to residues 892-917 (covering helices 2 and 27) and to residues 1392-1415 [34]. A further piece of evidence is the observation made many years ago [38] that streptomycin inhibits the cleavage by colicin E3 of the 16S RNA at position 1493; as already discussed above, this position is in close proximity to the 14~qregion. Thus the streptomycin data form a link between helix 18, the region of helices 2 and 27, and the 1400-region. The relationship between streptomycin and ribosomal protein Si2 has recently been discussed in detail by Powers and Noller [33]. All that needs to be mentioned here is the fact that this protein gives a foot-print [29] on 16S RNA in helix 18 and in the region of helices 19 and 27 (fig 3), and also shows effects at nucleotides 1392-1396. Thus, just as with the antibiotic data, these results establish a connection between the 527 and 1400 regions. The tRNA foot-printing data present a similar picture. Most of the foot-print sites [ 11 ] are concentrated in the cleft region of the RNA model (fig l), and in some cases there is direct evidence for the juxtaposition of the individual foot-print sites, such as those at positions 693 and 794-795 which are connected by the intra-RNA cross-link I (fig 3), or those in the region of helix 44 which are connected by phylogenetically established tertiary interactions and by the intra-RNA cross-link III (fig 3, and see below). Similar or even more extensive inter-connections between the corresponding tRNA foot-print sites in 23S RNA [39] have also been noted [40, 411. Again,
however, the data provide a link between the 527 and 1400 regions, with P-site bound tRNA showing footprints at positions 532 and 1399-1401 11 ~], and A-site bound tRNA showing foot-prints at positions 529531, 1408 and 1492-1493 [11]. Furthermore, the antibiotic neomycin mimics the A-site tRNA, showing foot-print effects at positions 525, 1408 and 1494 [9]. Although it is difficult to formally exclude the possibility that the effects observed in the 530 loop are allosteric, it seems highly unlikely that such different ligands as tRNA, protein S12, and two antibit~tics should all be able to cause allosteric effects at similar but not identical sites (a common conformational change induced by all the ligands would rather be expected to show effects at identical positions). The fourth line of evidence comes from sitedirected cross-linking experiments with mRNA [15], where an mRNA analogue carrying a thio-uridine residue at position +11 (relative to the 5'-base of the P-site codon) showed concomitant cross-linking to positions 532 and to positions 1392-1398, (the latter positions also having been observed in older crosslinking studies with a simple poly(A) messenger analogue [10], see fig 1). Here, it could be argued that two distinct binding sites for the mRNA were simultaneously involved, but, taken together with the data described above, this result provides additional circumstantial evidence for a juxtaposition of the 527 and 1400 regions. At all events, it seems dear that the evidence in favour of this juxtaposition now outweighs the somewhat self-contradictory electron microscopic evidence for a physical separation of the two areas, as discussed in the previous section.
The location of helix 34 If, as just suggested, the loop-end of helix 18 is to be relocated at the decoding site of the RNA model, then this would only leave the functional sites that have been observed in helix 34 on the 'wrong' side of the 30S subunit (fig 1). Helix 34 (fig 3) contains a site of resistance to the antibiotic spectinomycin [35], as well as foot-print sites to the latter [9] and a modified nucleotide (see below). However, there is now direct evidence that helix 34 lies close to the decoding site, again from site-directed cross-linking studies with mRNA analogues [37]. In this case, a thio-uridine residue located at position +6 relative to the 5'-base of the P-site codon in the mRNA was found to be involved in a tRNA-dependent cross-link to position ca 1055 of the 16S RNA in the 30S initiation complex. The position of the cross-link has since been precisely localized to position 1052, and has also been observed in the 70S initiation complex (Olga Dontsova, personal communication). Position +6 corre-
324 sponds to the 3'-base of the A-site codon, and this result thus places position 1052 in helix 34 directly in the decoding site of the 30S subunit. It should also be noted that this cross-link to helix 34 is only five bases in the mRNA away from that at the +11 position described in the previous section, which was crosslinked to position 532 of the 16S RNA. Helix 34 has been implicated in the termination event of protein synthesis, by the finding that a deletion at position 1054 causes suppression of the UGA termination signal 1421, and a model was proposed involving direct base-pairing between the UGA triplet and two tandem complementary UCA sequences in the opposing strand of helix 34. It now appears that in fact this deletion causes read-through of all three termination triplets, which casts doubt on the base-pairing model (Catherine Prescott, personal communication), but at the same time indicates a more universal involvement of helix 34 in termination. Since a base change in yeast mitochondria at a site corresponding to position 517 in E coli 16S RNA has been shown to cause supression of an ochre mutation [431, this provides a further circumstantial link between the 530 region and helix 34, which, as just described, must now be relocated to the decoding site of the 30S subunit.
The environment of helix 44, and clustering of modified nucleotides The location of helix 44, which is adjacent to the 1400 region of the 16S RNA (fig 3), is clearly relevant to any discussion of the topography of the ribosomal decoding site. Since publication of the RNA model [1 ], a number of observations have been made conceming the environment of this helix, some of which have already been mentioned in the preceding sections. First, the phylogenetically conserved tertiary interactions that have been reported between bases 1399 and 1495, 1401 and 1501, and 1405 and 1504 I221, impose important constraints on the singlestranded regions of RNA on either side of helix 44, and further confirmation of these constraints is provided by the intra-RNA cross-link III ([471; fig 3). Another intra-RNA cross-link has also been observed in this area [47] between positions 1393-1401 and 1531-1542 (cross-link II, fig 3), which effectively joins the extreme 3'-terminus of the 16S RNA with the 1400 region. This cross-link most probably reflects a flexibility of the T-terminus, which we have previously commented on [ 1], and raises a note of caution as to the placement of the T-terminus by electron microscopy [eg 44] as well as to the interpretation of its involvement in cross-links to mRNA analogues at positions upstream of the coding triplet [36].
Most important, however, is the very recent finding that bases 1408-1411 (at the left-hand end of helix 44, fig 3) are involved in a direct cross-link to 23S RNA at the 30S-50S subunit interface [48]. This indicates that helix 44 is incorrectly placedin the model (fig 1), and that at least its upper end should be relocated on the interface side of the 30S subunits, as opposed to the solvent side. Such a placement would bring the upper end of helix 44 (bases 1409/1491) close to the loop-end of helix 45 (see fig 1; the loop-end of this latter helix points downward in the model), and in fact this loop-end (an oligonucleotide covering positions 1518-1523) was also cross-linked to 23S RNA [48] at a site very close to or identical with that cross-linked to positions 1408-1411 of the 16S. Other recent observations support a location of helix 44 at the interface [48]. This raises the interesting question of the clustering of the modified nucleotides in the 16S and 23S RNA at the functional centres of the two subunits, which now appears to be much more extreme than hitherto supposed [I, 10]. In the 16S RNA, cross-link Ill and the tertiary interactions bring the three modified bases at positions 1402, 1407 and 1498 into close proximity with one another, and the interface cross-links just mentioned bring the further three modified bases at the loop-end of helix 45 (positions 1516, 1518 and 1519) into the same near neighbourhood. The two modified nucleotides at the loop-end of helix 31 (positions 966, 967) also appear to lie in the cleft of the subunit (fig 1), and - if the arguments presented in the previous sections are correct - then the remaining two modified nucleotides at position 527 (in the loop-end of helix 18) and position 1207 (in helix 34) also belonl~ to this cluster. Thus, all of the modified bases in the 16S RNA would then lie in the immediate vicinity of the decoding site. The situation in the 23S RNA is very similar. Nineteen modified nucleotides have now been identified in the E coli 23S RNA molecule (Cooperman B, Mitchell P, in preparation), and no less than eighteen of these are at positions which are either connected by direct intra-RNA cross-links [40, 41] or lie close to tRNA foot-print sites [39]. (The nineteenth, at position 1608 of the 23S RNA sequence, lies in a region which is so far devoid of topographical information, and thus may or may not also belong to the cluster.) Most dramatic, however, is the information provided by the interface cross-links discussed above; the site in the 23S RNA involved in these cross-links covers positions 1911-1920, where no less than three of the modified bases are located. Furthermore, both interface cross-links (to positions 14081411 and positions 1518-1523 of the 16S RNA) were also observed when polysomes were used as the substrate for the cross-linking reaction. Thus, the vast
325
majority, if not all, of the modified nucleotides in 16S and 23S RNA form a tight topographical cluster which is directly related to the binding site of tRNA and forms the active centre of the ribosome. A substantial proportion of the invariant bases in the 16S and 23S RNA also contribute to this cluster [48].
6 7 8 9
Conclusions The published three-dimensional models of the 16S ribosomal RNA [1, 7] have relied heavily on the results of immunoelectron microscopic studies (eg [41) and on data concerning the arrangement of the ribosomal proteins - in particular the neutron scattering map [3] - in order to locate the various RNA helices in the structure. However, because so little is known about the sizes and shapes of the individual proteins, the placement of an element of the RNA structure in the model on the basis of the corresponding position of a particular protein is subject to a large inherent error, and the resolution provided by the electron microscopic approach is similarly low. The arguments presented here suggest that in consequence several helices in the published models are incorrectly located, but some of the evidence for this is still circumstantial, being based on foot-printing type data, rather than on unambiguously direct observations and measurements. A further exploitation of the various cross-linking approaches, in particular site-directed cross-linking of functional ligands such as mRNA [15, 37] or tRNA [45, 46] as well as the precise analysis of intra-RNA cross-links within the 16S and 23S molecules themselves (eg [411), seems to offer the most promising approach for finally resolving these discrepancies and for developing the next generation of three-dimensional models for the ribosomal RNA.
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