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Journal of Biomolecular Structure and Dynamics Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tbsd20
Three-Dimensional Folding of Tetrahymena Thermophila rRNA IVS Sequence: A Proposal a
Giorgio Benedetti & Stefano Morosetti
a
a
Department of Chemistry , University of Rome I , P.le A. Moro 5, 00185 , Rome , Italy Published online: 21 May 2012.
To cite this article: Giorgio Benedetti & Stefano Morosetti (1991) ThreeDimensional Folding of Tetrahymena Thermophila rRNA IVS Sequence: A Proposal, Journal of Biomolecular Structure and Dynamics, 8:5, 1045-1055, DOI: 10.1080/07391102.1991.10507864 To link to this article: http://dx.doi.org/10.1080/07391102.1991.10507864
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is
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Journal of Biomolecular Structure & Dynamics, ISSN 0739-1102 Volume 8. l!sue Number 5 (1991). '"Adenine Press (1991).
Three-Dimensional Folding of Tetrahymena Thermophila rRNA IVS Sequence: A Proposal
Downloaded by [New York University] at 22:46 13 May 2015
Giorgio Benedetti and Stefano Morosetti Department of Chemistry University of Rome I P.le A Mora 5 00185 Rome, Italy Abstract We studied the Tetrahymena thermophila rRNA IVS sequence with the aim of obtaining a model of the structure characterized by the bases proximity of the self-reactions sites. The considered sequence kept up those fragments essential for its catalytic activity as demonstrated by deletion mutants. The first step was the theoretical analysis with a computer method previously proposed, to find optimal free energy secondary structures with the required features, under the suitable constrains. Then we tried folding the obtained secondary structures, in low resolution tertiary models, which kept up the proximity of the catalytic sites also in the space. The proposed tertiary folding seems to provide for a better explanation to the transesterification mechanisms and moreover it is in good agreement with the experimental data (activity of mutants, enzymatic cleavages, phylogenetically conserved regions).
Introduction Tetrahymena thermophila rRNAIVS sequence(IVS) has been widely studied for its ability in self-splicing and enzymatic activity ( 1). RNA self-splicing has been identified as a property of a number of other in trans found in precursor RNA from different organisms (2). This behavior can be important in the field of biotechnological applications (3) and it can suggest new hypotheses on the origin of life (4). The tertiary structure is essential in the mechanism of those reactions. Indeed activity is lost in denaturant conditions. Furthermore, deletions and a variety of bases substitutions affect the activity ( 1). To go inside the molecular mechanism of such reactions, it would be essential the knowledge of the three-dimensional folding of the molecule. The structure determination could be via X-ray crystallography or NMR spectrometry. However the IVS has not yet been crystallized, and the size of the molecule makes difficult to use these technics. In this situation it may be useful first to propose secondary foldings and then to try building three-dimensional models. The complexity of searching secondary structures requires the use of a computer method based on a theoretical approach able to take into account also the large quantity of experimental data available for this sequence at this moment. A tertiary model has already been proposed (5) but it shows the same limitation of the secondary
1045
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Benedetti & Morosetti Table I Deletion Mutants Retaining Self-Splicing Activity Deletion ~
5'exon -o- -10
~40-o-46
Reference 8 8
~56-;~ ~
~
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~
93 127-;- 195 225-;- 244 332-;- 409 +27 -o- 3'exon
12 9 II 8
structure from which it is derived (6): the self-splicing sites are not in close position to react. Here we show the proposal of a tertiary folding based on secondary structures obtained by our algorithm previously developed (7). It was used in an extensive analysis ofiVS sequence so as to obtain structures characterized by close proximity of self-reaction sites, a good agreement with the experimental data and low free energy content.
Methods Search for Secondary Structures
Reported deletion analyses point out the regions of the IVS sequence and adjacent exons, not important for self-splicing and cyclization activity (1,8-12). Our aim was to find the minimal structural core needed so that the splicing reaction works. Therefore, we studied a shortened sequence (see Table I) where all the deletions. which retain the self-splicing activity, are considered. We think that this strategy makes easier the search for the minimal tertiary structure able to self-splice. This choice was supported by the fact that the retained regions are generally involved in phylogenetically conserved base paired elements, which characterize group I IVS (13). This sequence was analyzed for obtaining secondary structures by our computer method previously developed (7,14). It is particularly apt for this kind of search, because its main features: the ability in addressing the search towards optimal free energy secondary structures characterized by the model hypothesis imposed (in this case the proximity of the self-splicing sites), and in keeping into account the experimental data in a gradual manner. This flexibility allows us to reach the best arrangement among the experimental data respect, the model hypothesis and the free energy content. It is obtained searching for structures with minimum T:
Footnote to Table II: Pi. i-th pairing element following the standard nomenclature for the group I IVS (24). Pi(5'), 5' segment of helix Pi; Pi(3'], 3' segment of helix Pi. P, Q. RandS are conserved internal
sequence elements characteristic of group I introns. Intron sequences are designed with upper-case letters; exon sequences with lower-case letters. Result: - no activity; -I+ weak activity; + wild type activity; + + activity greater than wild type.
1047
Three-Dimensional Folding of rRNA IVS Sequence Table II Splicing Activity of IVS Mutants
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Sequence Element P1[5'] Pl(5'] P1[5'] Pl(5'] Pl[3'] Pl[3'] Pl[3'] Pl(3'] Pl[3'] Pl[3'] Pl[3'] Pl(3'] Pl[3'] PI PI PI PI PI P3[5'] P3[3'] P3[3'] P3
Q
p P6[5'] P6[3'] P6 R R R R R R
s s s s
P7 P7 P8[5'] P8[3'] P8 P9.0[5'] P9.0[5'] P9.0(5'] P9.0[3'] P9.0 P10[5'] P10(3'] P10[3'] PI0(3'] P10(3'] PI0(3'] P10 3'exon 3'exon
Mutation Sites u-3a:c-2g u-lc u-la u-lg G23C G23A G23U G23C:A24U A24C G25C G26C G27C A28C c-4u/G25A c-2g/G23C u-3g/A24C c-4g/G25C u-3a:c-2g/G23C:A24U GIOOC:CI02U G272A:C274G U273C GIOOC:CI02U/G272A:C274G G212C C109U G215C:C216A G257U:U258G G215C:C216NG257U:U258G U259A:A261C C260A C260U C260G C266G:A268C C266G:A268U G309A U307G:G309C U307A:G309C A30 I C:A302G:G 303C C266G:A268C/U307G:G309C C266G :A268U/U 307 A:G 309C G280C:G282U C296A:C298G G280C:G282U/C296A:C298G A314C A314U G313U:A314U U412A:C413A G313U:A314U/U412A:C413A AI6G:CI7U:CI8G G414U G414C C413A C413G g+4c:g+5a:u+6c AI6G:CI7U:CI8G/g+4c:g+5a:u+6c u+lg a+2c
Result
-I+ -I+ -I+ -I+
+ ++ + + + + + +
+ +
-I+ -I+ -I+ -I+ -I+ -I+
++
+ + +
-I+ -I+ + +
-I+ -I+ + +
-I+ -I+
Reference 15 13 13 13 13 13 13 15 13 13 13 13 13 13 13 13 13 16 17 17 I 17 10 10 18 18 18 18 19 19 19 20 17 10 20 20 17 20 17 18 18 18 I I 21 21 21 22 23 23 23 23 22 22 23 23
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Benedetti & Morosetti
where .£\G 101 is the free energy content; S101 and ~ot are the experimental and model hypothesis contributions respectively;~ and A. the related coefficients (see references 7 and 14 for further details). In the last years a lot of mutagenic experiments have been conducted to examine the
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role of several parts of the core structure in self-splicing ofTetrahymena IVS. They are summarized in Table II. Data on nuclease accessibility of the intact IVS RNA are available (6); they can characterize the single and double strand regions. The experimental data regard the native sequence, but we think that they are valid also for the shortened sequence because it retains the catalytic activity and then likely the same catalytic core. In our searching for secondary structures computer program we account for the different experimental evidences in the following manner:
a Figure l: a) Secondary structure of Tetrahymena thermophila IVS and connected exons rRNA. The arrows indicate the self-splicing sites; bh.ck d indicate the positions of the considered deletions; • ends of deletion; e mutations sites. Only some bases are considered for clarity.--- joins bases near in the space but not in the bidimensional drawing. For other regards see Table II.
Three-Dimensional Folding of rRNA IVS Sequence
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a) the compensatory mutations can be considered as certain evidences of the existence of base pairings and therefore they are introduced as model hypotheses;
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b) in contrast the enzymatic cleavages are not completely reliable (25) and therefore they are introduced in a "soft" manner as experimental data. An experimental coefficient value As = 0.5 is the best arrangement between free energy and experimental data for obtaining the most structural variability into the set of optimal secondary foldings; bigger or smaller As values result in the prevalence of a factor over the other one and therefore in a limited variability; c) C260 base is imposed as single strand by model hypothesis because it is the more probable GTP binding site (19). Our aim was to find structures characterized by the self-reaction sites in close proximity, therefore we classified the obtained foldings on the basis of relative positions
PS P4
P9
b Figure I: b) Perspective view of our proposal of tertiary folding.
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Benedetti & Morosetti
of2, 16 and 414 bases. Three main goups can be found in the search for a set of 1000 final secondary structures. The first group is similar to the Cech's proposal (6); the other ones are characterized by the proximity of the self-splicing sites, even though free energy content and agreement with the experimental data are comparable with the first one. the second group of structures is of the kind already reported in our previous work (7); in particular they have 16 and 414 bases into two helical streams joined together (see Figure 2). The third group is characterized by the position of the same bases on different helical regions but in close proximity in the secondary representation (see Figure la).
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Tertiary Folding The next step of our study was to carry out the tertiary folding using the deepest free energy secondary structure for each class. The first group was not analyzed because the tertiary model has already been proposed by Kim et al. (5). It should point out that this folding does not seem to have the catalytic sites in close proximity, as it happens in the secondary structure from which it derives. We realized the tertiary foldings by using a low resolution model following the Seeman's approach (26). This model retains the essential characteristics of the three-dimensional highresolution structure. This can be considered as a first "filter" in testing the feasibility of a tertiary folding in agreement with the experimental data and credible in regard to its biological activity. Moreover it is the first step to obtain a detailed model at atomic level. Most of the building rules were derived from consideration of the experimental structures of tRNA (27) and X-ray fiber diffraction (28) as it follows: a) RNA duplexes were assumed to have A-form RNA helix conformation; b) the sites directly or indirectly involved in self-splicing reaction (2, 414 and 260 bases) are located in convenient positions for transesterification reaction; c) the helical axes of parallel strands are 22 A distant (28); d) two helical regions joined by single strand(s) are coli nearly stacked when possible; otherwise they are placed in adjacent position following c) criterion; e) single bulged bases were stacked within helix; f) a distance of 6 A was allowed between two adjacent nucleotides in single strand
regions. Results
In Figure 2 a detail of the region .:ontaining 2 and 414 bases is shown. It can be seen by the three-dimensional schemes (top) that it is not possible to get the self-splicing sites in position apt to transesterification reaction, utilizing the above rules. Therefore
1051
Three-Dimensional Folding of rRNA IVS Sequence
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8
8
A
c
8
a
c
b
Figure 2: a) and b) are the two possible arrangements of the core of the second class of structures. Top: three-dimensional folding: bottom: bi-dimensional folding.
we have not further on considered this group of structures. In contrast we observed that structures ofthethirdgroup had the possibility to held the catalytic sites inclose proximity in tertiary folding. Therefore we realized the whole three-dimensional model of the best free energy structure of this group. The perspective view of the model is shown in Figure 1b and the stereoview is shown in Figure 3. Afterwards we present the most attractive features of the model. In Figure 4 the detail regarding the regions involved in the GTP binding (J6/7 and J7 /8) and the
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Benedetti & Morosetti
Figure 3: Stereoview of the proposed three-dimensional folding.
2-15 X
y Figure 4: Detail of the self-splicing sites and of the single strand regions involved in the self-splicing reaction. Ji.k indicates a single strand which joins together P; and Pi regions. The arrow represents the GTP and it starts from C260 base (GTP binding site) and ends at 5' splicing site.
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Three-Dimensional Folding of rRNA IVS Sequence
1053
self-splicing reaction (PI and P3), is shown. The C260 base is in position apt to have Watson-Crick binding with GTP molecule, so as to put it at a suitable distance for reacting with the 5' site and therefore starting the self-reaction. The 3' site, located in front of the 5' one, is available to continue the reaction. The distance in the model of the two self-splicing sites is approximately of 6 A and it is in agreement with theoretical quantum-mechanical investigations (29). The three-dimensional position of259-261 and 301-303 bases can give a good interpretation of the known experimental data. Indeed single nucleotide mutations of the highly conserved residue C260 are defective in splicing ( 19) and this effect can be explained by the impossibility of the GTP binding. The primary effect of the U259A:A261 C mutation is to increase GTP binding ( 17); this might reflect a stability change due to direct stacking interactions among 259,261 bases and GTP. Alternatively C261 mutation could compete with C260 so that part of the GTP is in a position sterically less favorable to the splicing reaction. A301C:A302G:G303C mutant shows a marked loss of self-splicing activity; however, hydrolysis at the 3' site proceeds normally (17). This result can be justified by the presence of the bases 301C and 303C, free to compete with the C260 and in a position more exposed to the environment. The alternative complex formation could cause a structural distortion that indirectly alters a structure required for transesterification. Another characteristic of our model in comparison with the other ones is a different arrangement of the 5' exon-intron junction. The proposed PI helical region is reorganized in the part not investigated by mutation experiments, to form a hairpin including 2-15 bases. This proposal has the advantage to put the 2-15 bases in a position sterically favorable to the following cyclization reaction ofiVS with the removal of 2-15 fragment. In the chain oftransesterification reactions, the single strand fragment 16-21 is available to work in two consecutive roles: a) to hold the 5' exon in a suitable position for the nucleophilic attack at the 3' splice site, by the PIO formation (30); b) to put the bases 15-16 in position to get the cyclization reaction by local rearrangement of the structure. On the other hand this hairpin is consistent with the enzymatic data indicating the presence of a double strand in this region (6). Finally in Figure 5 the P7 double strand and all the helical regions (P3, P6, P8 and P9) directly joined to it, are shown. It must be remarked the crucial position ofP7 in keeping the structure in a close-packed fashion. This appears to be consistent with the observation that the nucleotide sequence is very highly conserved in this region and the mutants experiments (17 ,20) suggest that the sequence ofP7 is important in splicing activity (I). In fact we expect that a relatively small double helix region surrounded by a large number of single strand regions, will be stabilized by tertiary interactions.
Conclusions Our aim was to obtain a tertiary structure model ofiVS and adjacent exons characterized by close proximity of self-reaction sites. We shortened the sequence to those parts necessary to self-splicing activity, as demonstrated by mutant deletion experiments. We studied this sequence in order to obtain a large set of optimal secondary
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Benedetti & Morosetti
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pg
l::y
X
z Figure 5: Detail of the structure regarding P7 and helical regions joined to it. The connecting single strand regions are in dark.
structures substantially satisfYing the available experimental data (enzymatic cleavages, mutants, phylogenetic evidences). The secondary structures were classified into subsets on the basis of the relative positions of the self-reaction sites (2, 15 and 414 bases). We attempted the tertiary folding of a representative secondary structure (the deepest free energy one) for each subset, to test the possibility to get the self-reaction sites in close proximity in space. The criteria utilized to realize a low resolution tertiary model, mainly derive from experimental X-ray structures (tRNA and fibers). Our tertiary structure presents the required close proximity of the reaction sites. It also shows other attractive features: 1) the C260 base is in a suitable position for getting a GTP molecule via Watson-Crick binding, in proximity of the 5' splicing site; 2) the 2-15 fragment is arranged in a helical form and in a proper position so as to facilitate its excision; 3) the 301-303 bases are located in a single strand in front of the catalytic center and therefore they can influence the reaction kinetics as indicated by mutagenic experiments; 4) the P7 has a central role in joining helixes together in a compact structure and this is in agreement with the importance of its pairing sequence as demonstrated by various experimental observations. We hope our proposal will be useful for design of specific experiments with the aim
Three-Dimensional Folding of rRNA IVS Sequence
1055
of a better understanding of self-reactions molecular mechanisms in the IVS of rRNA Tetrahymena thermophila. This model could be the first step to obtain a high resolution one, in particular the catalytic center at atomic level.
Acknowledgments This work has been supported by Ministero Pubblica Istruzione Grant 89 20902 337.
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References and Footnotes
I. 2. 3. 4. 5. 6. 7. 8. 9. 10. II. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27.
28. 29. 30.
Burke. J.M .. Gene 73.273-294 (1988). Cech. T.R.. Gene 73.259-271 (1988). Maddox, 1.. Nature342. 609-613 (1989). Gilbert, W .. Nature 319. 618 ( 1986). Kim. S.-H. and Cech. T.R.. Proc. Nat/. Acad. Sci. USA 84, 8788-8792 (1987). Cech. T.R.. Tanner. N.K.. Tinoco, !..Jr.. Weir. B.R.. Zucker. M. and Perlman. P.S .. Proc Nat/. Acad. Sci. USA 80. 3903-3907 ( 1983 ). Benedetti. G .. DeSantis. P. and Morosetti. S.. J Biomol. Struct. Dyn. 7, 1269-1277 (1990). Price, J.V.. Engberg. J. and Cech. T.R.. J Mol. Bioi. 196.49-60 ( 1987). Price. J.V .. Kieft. G.L., Kent. J.R.. Sievers. E.L. and Cech. T.R.. Nucleic Acids Research 13. 18711889 (1985). Waring. R.B .. Ray. J.A.. Eduars, S.W.. Scazzocchio. C. and Davies. R.W .. Ce/140. 371-380 (1985). Barfod. E.T. and Cech. T.R.. Genes Develop. 2. 652-663 ( 1988). Joyce, G.F .. van der Horst. G. and Inoue. T .. Nucleic Acids Research 17. 7879-7889 ( 1989). Been. M.D .. Barfod. E.T.. Burke. J.M .. Price. J.V .. Tanner. N.K., Zaug, A.J. and Cech. T.R.. Cold Spring Harbor Symp. Quant. Bioi. L/1. 147-157 (1987). Benedetti. G .. De San tis. P. and Morosetti. S.. Nucleic Acids Research 17. 5149-5161 (1989). Davis. R.W .. Waring, R.B. and Towner. P .. Cold Spring Harbor Symp. Quant. Bioi. L/1. 165-172 (1987). Waring, R.B .. Towner. P .. Minter, S.J. and Davis. R.W., Nature 321. 133-139 (1986). Williamson. C.L Tierney. W.M .. Kerker. B.J. and Burke. J.M .. J Bioi. Chern. 262, 14672-14682 (1987). Williamson. C.L Desai. N.M. and Burke, J.M .. Nucleic Acids Research 17.675-690 (1989). Yarus. M .• Levine. J .. Morin. G.B. and Cech. T.R., Nucleic Acids Research 17.6969-6981 (1989). Burke. J.M .. Irvine. K.D .. Kaneko. K.J .. Kerker. B.• Oettgen. A.B., Tierney, W.M .. Williamson. C.L Zaug. A.J. and Cech. T.R.. Ce/145. 167-176 (1986). Burke. J.M .. Esherick. K.S .. Burfeind. W.R. and King. J.L.. Nature 344. 80-82 (1990). Michel. F .. Hanna. M .. Green, R.. Bartel. D.P. and Szostak. J.W .. Nature 342.391-395 ( 1989). Price, J.V .. Cech. T.R.. Genes Develop. 2. 1439-1447 (1988). Burke, J.. Belfort. M .. Cech. T.R .. Davies. R.W .. Schweyn, R .. Shub. D .. Szostak, J. and Tabak. H .. Nucleic Acids Research 15. 7217-7221 (1987). Noller. H.F. and Woese. C.R., Science 212.403-411 (1981). Seeman, C.N ..J Biomol. Struct. Dyn. 5. 997-1004 (1988). Kim. S.-H .. in Tran.~fer RNA: Structure. Properties, and Recognition. Cold Spring Harbor Monograph Series 9A. Eds .. Schimmel. P .. Soli. D. and Abelson.J.. (Cold Spring Harbor Laboratory. Cold Spring Harbor, NY) 83-100 (1979). Arnott. S.. Progr. Biophys. Mol. Bioi. 21.267-319 (1970). McCourt, M .. Shibata. M .. Mciver. J.W .. Jr. and Rein, R., J Mol. Struct. (Theochem.) 179. 145-152 (1988). Davis. R.W .. Nature 300. 719-724 ( 1982).
Date Received: Sept. 6. 1990
Communicated by the Editor R.H. Sarma
Journal of Biomolecular Structure and Dynamics Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tbsd20
Three-Dimensional Folding of Tetrahymena Thermophila rRNA IVS Sequence: A Proposal a
Giorgio Benedetti & Stefano Morosetti
a
a
Department of Chemistry , University of Rome I , P.le A. Moro 5, 00185 , Rome , Italy Published online: 21 May 2012.
To cite this article: Giorgio Benedetti & Stefano Morosetti (1991) ThreeDimensional Folding of Tetrahymena Thermophila rRNA IVS Sequence: A Proposal, Journal of Biomolecular Structure and Dynamics, 8:5, 1045-1055, DOI: 10.1080/07391102.1991.10507864 To link to this article: http://dx.doi.org/10.1080/07391102.1991.10507864
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is
Downloaded by [New York University] at 22:46 13 May 2015
expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/terms-and-conditions
Journal of Biomolecular Structure & Dynamics, ISSN 0739-1102 Volume 8. l!sue Number 5 (1991). '"Adenine Press (1991).
Three-Dimensional Folding of Tetrahymena Thermophila rRNA IVS Sequence: A Proposal
Downloaded by [New York University] at 22:46 13 May 2015
Giorgio Benedetti and Stefano Morosetti Department of Chemistry University of Rome I P.le A Mora 5 00185 Rome, Italy Abstract We studied the Tetrahymena thermophila rRNA IVS sequence with the aim of obtaining a model of the structure characterized by the bases proximity of the self-reactions sites. The considered sequence kept up those fragments essential for its catalytic activity as demonstrated by deletion mutants. The first step was the theoretical analysis with a computer method previously proposed, to find optimal free energy secondary structures with the required features, under the suitable constrains. Then we tried folding the obtained secondary structures, in low resolution tertiary models, which kept up the proximity of the catalytic sites also in the space. The proposed tertiary folding seems to provide for a better explanation to the transesterification mechanisms and moreover it is in good agreement with the experimental data (activity of mutants, enzymatic cleavages, phylogenetically conserved regions).
Introduction Tetrahymena thermophila rRNAIVS sequence(IVS) has been widely studied for its ability in self-splicing and enzymatic activity ( 1). RNA self-splicing has been identified as a property of a number of other in trans found in precursor RNA from different organisms (2). This behavior can be important in the field of biotechnological applications (3) and it can suggest new hypotheses on the origin of life (4). The tertiary structure is essential in the mechanism of those reactions. Indeed activity is lost in denaturant conditions. Furthermore, deletions and a variety of bases substitutions affect the activity ( 1). To go inside the molecular mechanism of such reactions, it would be essential the knowledge of the three-dimensional folding of the molecule. The structure determination could be via X-ray crystallography or NMR spectrometry. However the IVS has not yet been crystallized, and the size of the molecule makes difficult to use these technics. In this situation it may be useful first to propose secondary foldings and then to try building three-dimensional models. The complexity of searching secondary structures requires the use of a computer method based on a theoretical approach able to take into account also the large quantity of experimental data available for this sequence at this moment. A tertiary model has already been proposed (5) but it shows the same limitation of the secondary
1045
1046
Benedetti & Morosetti Table I Deletion Mutants Retaining Self-Splicing Activity Deletion ~
5'exon -o- -10
~40-o-46
Reference 8 8
~56-;~ ~
~
Downloaded by [New York University] at 22:46 13 May 2015
~
93 127-;- 195 225-;- 244 332-;- 409 +27 -o- 3'exon
12 9 II 8
structure from which it is derived (6): the self-splicing sites are not in close position to react. Here we show the proposal of a tertiary folding based on secondary structures obtained by our algorithm previously developed (7). It was used in an extensive analysis ofiVS sequence so as to obtain structures characterized by close proximity of self-reaction sites, a good agreement with the experimental data and low free energy content.
Methods Search for Secondary Structures
Reported deletion analyses point out the regions of the IVS sequence and adjacent exons, not important for self-splicing and cyclization activity (1,8-12). Our aim was to find the minimal structural core needed so that the splicing reaction works. Therefore, we studied a shortened sequence (see Table I) where all the deletions. which retain the self-splicing activity, are considered. We think that this strategy makes easier the search for the minimal tertiary structure able to self-splice. This choice was supported by the fact that the retained regions are generally involved in phylogenetically conserved base paired elements, which characterize group I IVS (13). This sequence was analyzed for obtaining secondary structures by our computer method previously developed (7,14). It is particularly apt for this kind of search, because its main features: the ability in addressing the search towards optimal free energy secondary structures characterized by the model hypothesis imposed (in this case the proximity of the self-splicing sites), and in keeping into account the experimental data in a gradual manner. This flexibility allows us to reach the best arrangement among the experimental data respect, the model hypothesis and the free energy content. It is obtained searching for structures with minimum T:
Footnote to Table II: Pi. i-th pairing element following the standard nomenclature for the group I IVS (24). Pi(5'), 5' segment of helix Pi; Pi(3'], 3' segment of helix Pi. P, Q. RandS are conserved internal
sequence elements characteristic of group I introns. Intron sequences are designed with upper-case letters; exon sequences with lower-case letters. Result: - no activity; -I+ weak activity; + wild type activity; + + activity greater than wild type.
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Three-Dimensional Folding of rRNA IVS Sequence Table II Splicing Activity of IVS Mutants
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Sequence Element P1[5'] Pl(5'] P1[5'] Pl(5'] Pl[3'] Pl[3'] Pl[3'] Pl(3'] Pl[3'] Pl[3'] Pl[3'] Pl(3'] Pl[3'] PI PI PI PI PI P3[5'] P3[3'] P3[3'] P3
Q
p P6[5'] P6[3'] P6 R R R R R R
s s s s
P7 P7 P8[5'] P8[3'] P8 P9.0[5'] P9.0[5'] P9.0(5'] P9.0[3'] P9.0 P10[5'] P10(3'] P10[3'] PI0(3'] P10(3'] PI0(3'] P10 3'exon 3'exon
Mutation Sites u-3a:c-2g u-lc u-la u-lg G23C G23A G23U G23C:A24U A24C G25C G26C G27C A28C c-4u/G25A c-2g/G23C u-3g/A24C c-4g/G25C u-3a:c-2g/G23C:A24U GIOOC:CI02U G272A:C274G U273C GIOOC:CI02U/G272A:C274G G212C C109U G215C:C216A G257U:U258G G215C:C216NG257U:U258G U259A:A261C C260A C260U C260G C266G:A268C C266G:A268U G309A U307G:G309C U307A:G309C A30 I C:A302G:G 303C C266G:A268C/U307G:G309C C266G :A268U/U 307 A:G 309C G280C:G282U C296A:C298G G280C:G282U/C296A:C298G A314C A314U G313U:A314U U412A:C413A G313U:A314U/U412A:C413A AI6G:CI7U:CI8G G414U G414C C413A C413G g+4c:g+5a:u+6c AI6G:CI7U:CI8G/g+4c:g+5a:u+6c u+lg a+2c
Result
-I+ -I+ -I+ -I+
+ ++ + + + + + +
+ +
-I+ -I+ -I+ -I+ -I+ -I+
++
+ + +
-I+ -I+ + +
-I+ -I+ + +
-I+ -I+
Reference 15 13 13 13 13 13 13 15 13 13 13 13 13 13 13 13 13 16 17 17 I 17 10 10 18 18 18 18 19 19 19 20 17 10 20 20 17 20 17 18 18 18 I I 21 21 21 22 23 23 23 23 22 22 23 23
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where .£\G 101 is the free energy content; S101 and ~ot are the experimental and model hypothesis contributions respectively;~ and A. the related coefficients (see references 7 and 14 for further details). In the last years a lot of mutagenic experiments have been conducted to examine the
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role of several parts of the core structure in self-splicing ofTetrahymena IVS. They are summarized in Table II. Data on nuclease accessibility of the intact IVS RNA are available (6); they can characterize the single and double strand regions. The experimental data regard the native sequence, but we think that they are valid also for the shortened sequence because it retains the catalytic activity and then likely the same catalytic core. In our searching for secondary structures computer program we account for the different experimental evidences in the following manner:
a Figure l: a) Secondary structure of Tetrahymena thermophila IVS and connected exons rRNA. The arrows indicate the self-splicing sites; bh.ck d indicate the positions of the considered deletions; • ends of deletion; e mutations sites. Only some bases are considered for clarity.--- joins bases near in the space but not in the bidimensional drawing. For other regards see Table II.
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a) the compensatory mutations can be considered as certain evidences of the existence of base pairings and therefore they are introduced as model hypotheses;
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b) in contrast the enzymatic cleavages are not completely reliable (25) and therefore they are introduced in a "soft" manner as experimental data. An experimental coefficient value As = 0.5 is the best arrangement between free energy and experimental data for obtaining the most structural variability into the set of optimal secondary foldings; bigger or smaller As values result in the prevalence of a factor over the other one and therefore in a limited variability; c) C260 base is imposed as single strand by model hypothesis because it is the more probable GTP binding site (19). Our aim was to find structures characterized by the self-reaction sites in close proximity, therefore we classified the obtained foldings on the basis of relative positions
PS P4
P9
b Figure I: b) Perspective view of our proposal of tertiary folding.
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of2, 16 and 414 bases. Three main goups can be found in the search for a set of 1000 final secondary structures. The first group is similar to the Cech's proposal (6); the other ones are characterized by the proximity of the self-splicing sites, even though free energy content and agreement with the experimental data are comparable with the first one. the second group of structures is of the kind already reported in our previous work (7); in particular they have 16 and 414 bases into two helical streams joined together (see Figure 2). The third group is characterized by the position of the same bases on different helical regions but in close proximity in the secondary representation (see Figure la).
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Tertiary Folding The next step of our study was to carry out the tertiary folding using the deepest free energy secondary structure for each class. The first group was not analyzed because the tertiary model has already been proposed by Kim et al. (5). It should point out that this folding does not seem to have the catalytic sites in close proximity, as it happens in the secondary structure from which it derives. We realized the tertiary foldings by using a low resolution model following the Seeman's approach (26). This model retains the essential characteristics of the three-dimensional highresolution structure. This can be considered as a first "filter" in testing the feasibility of a tertiary folding in agreement with the experimental data and credible in regard to its biological activity. Moreover it is the first step to obtain a detailed model at atomic level. Most of the building rules were derived from consideration of the experimental structures of tRNA (27) and X-ray fiber diffraction (28) as it follows: a) RNA duplexes were assumed to have A-form RNA helix conformation; b) the sites directly or indirectly involved in self-splicing reaction (2, 414 and 260 bases) are located in convenient positions for transesterification reaction; c) the helical axes of parallel strands are 22 A distant (28); d) two helical regions joined by single strand(s) are coli nearly stacked when possible; otherwise they are placed in adjacent position following c) criterion; e) single bulged bases were stacked within helix; f) a distance of 6 A was allowed between two adjacent nucleotides in single strand
regions. Results
In Figure 2 a detail of the region .:ontaining 2 and 414 bases is shown. It can be seen by the three-dimensional schemes (top) that it is not possible to get the self-splicing sites in position apt to transesterification reaction, utilizing the above rules. Therefore
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8
8
A
c
8
a
c
b
Figure 2: a) and b) are the two possible arrangements of the core of the second class of structures. Top: three-dimensional folding: bottom: bi-dimensional folding.
we have not further on considered this group of structures. In contrast we observed that structures ofthethirdgroup had the possibility to held the catalytic sites inclose proximity in tertiary folding. Therefore we realized the whole three-dimensional model of the best free energy structure of this group. The perspective view of the model is shown in Figure 1b and the stereoview is shown in Figure 3. Afterwards we present the most attractive features of the model. In Figure 4 the detail regarding the regions involved in the GTP binding (J6/7 and J7 /8) and the
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Figure 3: Stereoview of the proposed three-dimensional folding.
2-15 X
y Figure 4: Detail of the self-splicing sites and of the single strand regions involved in the self-splicing reaction. Ji.k indicates a single strand which joins together P; and Pi regions. The arrow represents the GTP and it starts from C260 base (GTP binding site) and ends at 5' splicing site.
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self-splicing reaction (PI and P3), is shown. The C260 base is in position apt to have Watson-Crick binding with GTP molecule, so as to put it at a suitable distance for reacting with the 5' site and therefore starting the self-reaction. The 3' site, located in front of the 5' one, is available to continue the reaction. The distance in the model of the two self-splicing sites is approximately of 6 A and it is in agreement with theoretical quantum-mechanical investigations (29). The three-dimensional position of259-261 and 301-303 bases can give a good interpretation of the known experimental data. Indeed single nucleotide mutations of the highly conserved residue C260 are defective in splicing ( 19) and this effect can be explained by the impossibility of the GTP binding. The primary effect of the U259A:A261 C mutation is to increase GTP binding ( 17); this might reflect a stability change due to direct stacking interactions among 259,261 bases and GTP. Alternatively C261 mutation could compete with C260 so that part of the GTP is in a position sterically less favorable to the splicing reaction. A301C:A302G:G303C mutant shows a marked loss of self-splicing activity; however, hydrolysis at the 3' site proceeds normally (17). This result can be justified by the presence of the bases 301C and 303C, free to compete with the C260 and in a position more exposed to the environment. The alternative complex formation could cause a structural distortion that indirectly alters a structure required for transesterification. Another characteristic of our model in comparison with the other ones is a different arrangement of the 5' exon-intron junction. The proposed PI helical region is reorganized in the part not investigated by mutation experiments, to form a hairpin including 2-15 bases. This proposal has the advantage to put the 2-15 bases in a position sterically favorable to the following cyclization reaction ofiVS with the removal of 2-15 fragment. In the chain oftransesterification reactions, the single strand fragment 16-21 is available to work in two consecutive roles: a) to hold the 5' exon in a suitable position for the nucleophilic attack at the 3' splice site, by the PIO formation (30); b) to put the bases 15-16 in position to get the cyclization reaction by local rearrangement of the structure. On the other hand this hairpin is consistent with the enzymatic data indicating the presence of a double strand in this region (6). Finally in Figure 5 the P7 double strand and all the helical regions (P3, P6, P8 and P9) directly joined to it, are shown. It must be remarked the crucial position ofP7 in keeping the structure in a close-packed fashion. This appears to be consistent with the observation that the nucleotide sequence is very highly conserved in this region and the mutants experiments (17 ,20) suggest that the sequence ofP7 is important in splicing activity (I). In fact we expect that a relatively small double helix region surrounded by a large number of single strand regions, will be stabilized by tertiary interactions.
Conclusions Our aim was to obtain a tertiary structure model ofiVS and adjacent exons characterized by close proximity of self-reaction sites. We shortened the sequence to those parts necessary to self-splicing activity, as demonstrated by mutant deletion experiments. We studied this sequence in order to obtain a large set of optimal secondary
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pg
l::y
X
z Figure 5: Detail of the structure regarding P7 and helical regions joined to it. The connecting single strand regions are in dark.
structures substantially satisfYing the available experimental data (enzymatic cleavages, mutants, phylogenetic evidences). The secondary structures were classified into subsets on the basis of the relative positions of the self-reaction sites (2, 15 and 414 bases). We attempted the tertiary folding of a representative secondary structure (the deepest free energy one) for each subset, to test the possibility to get the self-reaction sites in close proximity in space. The criteria utilized to realize a low resolution tertiary model, mainly derive from experimental X-ray structures (tRNA and fibers). Our tertiary structure presents the required close proximity of the reaction sites. It also shows other attractive features: 1) the C260 base is in a suitable position for getting a GTP molecule via Watson-Crick binding, in proximity of the 5' splicing site; 2) the 2-15 fragment is arranged in a helical form and in a proper position so as to facilitate its excision; 3) the 301-303 bases are located in a single strand in front of the catalytic center and therefore they can influence the reaction kinetics as indicated by mutagenic experiments; 4) the P7 has a central role in joining helixes together in a compact structure and this is in agreement with the importance of its pairing sequence as demonstrated by various experimental observations. We hope our proposal will be useful for design of specific experiments with the aim
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of a better understanding of self-reactions molecular mechanisms in the IVS of rRNA Tetrahymena thermophila. This model could be the first step to obtain a high resolution one, in particular the catalytic center at atomic level.
Acknowledgments This work has been supported by Ministero Pubblica Istruzione Grant 89 20902 337.
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References and Footnotes
I. 2. 3. 4. 5. 6. 7. 8. 9. 10. II. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27.
28. 29. 30.
Burke. J.M .. Gene 73.273-294 (1988). Cech. T.R.. Gene 73.259-271 (1988). Maddox, 1.. Nature342. 609-613 (1989). Gilbert, W .. Nature 319. 618 ( 1986). Kim. S.-H. and Cech. T.R.. Proc. Nat/. Acad. Sci. USA 84, 8788-8792 (1987). Cech. T.R.. Tanner. N.K.. Tinoco, !..Jr.. Weir. B.R.. Zucker. M. and Perlman. P.S .. Proc Nat/. Acad. Sci. USA 80. 3903-3907 ( 1983 ). Benedetti. G .. DeSantis. P. and Morosetti. S.. J Biomol. Struct. Dyn. 7, 1269-1277 (1990). Price, J.V.. Engberg. J. and Cech. T.R.. J Mol. Bioi. 196.49-60 ( 1987). Price. J.V .. Kieft. G.L., Kent. J.R.. Sievers. E.L. and Cech. T.R.. Nucleic Acids Research 13. 18711889 (1985). Waring. R.B .. Ray. J.A.. Eduars, S.W.. Scazzocchio. C. and Davies. R.W .. Ce/140. 371-380 (1985). Barfod. E.T. and Cech. T.R.. Genes Develop. 2. 652-663 ( 1988). Joyce, G.F .. van der Horst. G. and Inoue. T .. Nucleic Acids Research 17. 7879-7889 ( 1989). Been. M.D .. Barfod. E.T.. Burke. J.M .. Price. J.V .. Tanner. N.K., Zaug, A.J. and Cech. T.R.. Cold Spring Harbor Symp. Quant. Bioi. L/1. 147-157 (1987). Benedetti. G .. De San tis. P. and Morosetti. S.. Nucleic Acids Research 17. 5149-5161 (1989). Davis. R.W .. Waring, R.B. and Towner. P .. Cold Spring Harbor Symp. Quant. Bioi. L/1. 165-172 (1987). Waring, R.B .. Towner. P .. Minter, S.J. and Davis. R.W., Nature 321. 133-139 (1986). Williamson. C.L Tierney. W.M .. Kerker. B.J. and Burke. J.M .. J Bioi. Chern. 262, 14672-14682 (1987). Williamson. C.L Desai. N.M. and Burke, J.M .. Nucleic Acids Research 17.675-690 (1989). Yarus. M .• Levine. J .. Morin. G.B. and Cech. T.R., Nucleic Acids Research 17.6969-6981 (1989). Burke. J.M .. Irvine. K.D .. Kaneko. K.J .. Kerker. B.• Oettgen. A.B., Tierney, W.M .. Williamson. C.L Zaug. A.J. and Cech. T.R.. Ce/145. 167-176 (1986). Burke. J.M .. Esherick. K.S .. Burfeind. W.R. and King. J.L.. Nature 344. 80-82 (1990). Michel. F .. Hanna. M .. Green, R.. Bartel. D.P. and Szostak. J.W .. Nature 342.391-395 ( 1989). Price, J.V .. Cech. T.R.. Genes Develop. 2. 1439-1447 (1988). Burke, J.. Belfort. M .. Cech. T.R .. Davies. R.W .. Schweyn, R .. Shub. D .. Szostak, J. and Tabak. H .. Nucleic Acids Research 15. 7217-7221 (1987). Noller. H.F. and Woese. C.R., Science 212.403-411 (1981). Seeman, C.N ..J Biomol. Struct. Dyn. 5. 997-1004 (1988). Kim. S.-H .. in Tran.~fer RNA: Structure. Properties, and Recognition. Cold Spring Harbor Monograph Series 9A. Eds .. Schimmel. P .. Soli. D. and Abelson.J.. (Cold Spring Harbor Laboratory. Cold Spring Harbor, NY) 83-100 (1979). Arnott. S.. Progr. Biophys. Mol. Bioi. 21.267-319 (1970). McCourt, M .. Shibata. M .. Mciver. J.W .. Jr. and Rein, R., J Mol. Struct. (Theochem.) 179. 145-152 (1988). Davis. R.W .. Nature 300. 719-724 ( 1982).
Date Received: Sept. 6. 1990
Communicated by the Editor R.H. Sarma
