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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
Translation-Initiation Promoting Site on Transcripts of Highly Expressed Genes From Saccharomyces cerevisiae and the Role of Hairpin Stems to Position the Site Near the Initiation Codon a
T. A. Thanaraj & M. W. Pandit
a
a
Centre for Cellular and Molecular Biology , Hyderabad , 500 007 , India Published online: 21 May 2012.
To cite this article: T. A. Thanaraj & M. W. Pandit (1990) Translation-Initiation Promoting Site on Transcripts of Highly Expressed Genes From Saccharomyces cerevisiae and the Role of Hairpin Stems to Position the Site Near the Initiation Codon, Journal of Biomolecular Structure and Dynamics, 7:6, 1279-1289, DOI: 10.1080/07391102.1990.10508565 To link to this article: http://dx.doi.org/10.1080/07391102.1990.10508565
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.
Downloaded by [Rutgers University] at 22:23 08 April 2015
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 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, /SSN 0739-1102 Volume 7, Issue Number 6 (1990), "'Adenine Press (1990).
Translation-Initiation Promoting Site on Transcripts of Highly Expressed Genes From Saccharomyces cerevisiae and the Role of Hairpin Stems to Position the Site Near the Initiation Codon Downloaded by [Rutgers University] at 22:23 08 April 2015
T.A. Thanaraj and M.W. Pandit* Centre for Cellular and Molecular Biology Hyderabad 500 007, India Abstract It is reported that the AUG-upstream region on mRNAs of highly expressed genes from S. cerevisiae invariably possesses a translation-initiation promoting site, that can base pair with a well-conserved site on 18S rRNA during the formation of 40S initiation complex. Weak hairpin stem in the mRNA region between such a site and the initiation codon brings the site nearer to the initiation codon and also extends the length of base pairing. Such a base pairing can lead to a comparatively more stable 40S initiation complex, resulting in a higher rate of formation of the 80S initiation complex and consequently in an enhancement ofthe rate of initiation of translation. The site on 18S rRNA can interchange its base pairing between the site on mRNA and a well-conserved site on 25S rRNA in the formation of the 80S initiation complex.
Introduction The differential levels of the cellular proteins in eukaryotes can be attributed to the controls due to the various steps involved in the pathway from gene to protein, the major ones being mRNA transcription, processing, transport to the cytoplasm, and translation. The turnover rates of the mRNAs and proteins also play a role to this effect. There is much evidence to support the view that a significant degree of translational control occurs in eukaryotes (1,2). The controls for translational efficiency are exerted through processes such as initiation complex formation, chain elongation and termination. Even though the translational efficiency may be influenced by the level of elongation (3,4), the primary controlling factor is the rate of initiation (1,5-7). The scanning model for translation in eukaryotes as proposed and updated recently by Kozak (8) implies that the small ribosomal subunit binds initially at/near the 5' cap structure of the mRNA and then migrates along the mRNA to stop at the first AUG codon that is in a favourable context for initiating translation. Since every eukaryotic mRNA is *Author to whom correspondence should be addressed.
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capped, the difference in the rate of initiation must be governed by factors other than the cap structure. Secondary structure of mRNA in the 5' region has been shown to play a role (9-12). Consensus sequences in the AUG-context, which can act as signals for recognition of the functional AUG codon, have also been identified for both yeast and mammalian mRNAs (13,14). Mutations in the AUG-context sequence (5'-CC~CCAUGG-3') of mammalian mRNAs modulate the levels of translation-initiation to the extent of >20-fold (14). The mutations in the AUGcontext sequence (5'{AtAAUGU-3') of yeast mRNAs modulate the translationinitiation efficiency up to 2-fold (12), even though the extent of such a modulation is not as high as in the case of mammalian mRNAs. Cigan and Donahue (13) could identify differences in base composition of yeast leader regions- highly and lowly expressed genes having A- and G-richness, respectively. Leader-shuffiing experiments have documented the ability of certain cellular and viral 5' sequences to support efficient translation (8). Even though the AUG-context sequence in mammals has been proposed to base pair with a motif on 18S rRNA(l4), the role of mRNA-rRNA base pairing interactions in the scanning model have not been well characterised. The role of such a base pairing which may enhance the stability of the 40S initiation complex cannot be ruled out. Highly stable 40S initiation complex will lead to a higher rate of formation of 80S initiation complex leading to the enhanced translational yield. The involvement of the 3' end fragment (corresponding to the colicin fragment in E. coli) ofrRNA from the small subunit, in the initiation of translation is sufficiently well established in prokaryotes. The 3' end fragment of 16S rRNAfromE. coli contains the anti -Shine-Dalgamo sequence. Recently, we have reported sufficient suggestive evidence for the involvement of the hexamer (5'-GGAUCA-3'), from the abovementioned fragment, in the promotion ofthe translation-initiation by virtue ofbase pairing with a translation-initiation promoting (TP) site on mRNAs of highly expressed genes from E. coli (15). The 3' end fragment of the small subunit rRNA from E. coli as well as S. cerevisiae is shown in Figure 1. In mammalian cells, the involvement of the motif 5'-GGUGG-3' in base pairing with the AUG-context G A
G A G
c
G
A
G C A U U • G G c
g g A A
:
3'
I
A U U
U G G A
U G C C
c a ~s c D
u u aTP c
5'-ACAAggy _____ ggAUCA
~
S •- ACAAGGU
A C C U
G C G G
~ aTP G"iiifiCAUUA-3'
Extended a.TP
.S. cereuisiae Figure 1: Central hairpin stem of the 3' end fragment of the small subunit rRNAs. The site equivalent to the anti-(AUG-context) sequence of mammalian rRNA is indicated (dashed line). Watson-Crick base pairing is shown by colon(:); A· G and G · U by small circle. aTP, anti-TP site; aSD, anti-SD site.
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Translation-Initiation Promoting Site
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sequence has been suggested (14). Such a motif comprises of subsequences 5'-GGU-3' and 5'-GG-3' (shown by dashed line in Figure 1) held together by the central hairpin stem of the 3' end fragment of the 18S rRNA The 3'legofthe central hairpin stem is particularly interesting. In E. coli, as shown in Figure 1, this leg is formed by the sequence 5'-GGAUCACCUCCUUA-3', which consists of anti-SD (aSD) region flanked by an additional hexamer (designated as aTP) and the 3' terminal bases. In archaebacteria and chloroplast, this pattern as well as the individual bases therein are well conserved. In eukaryotes, the anti-SD region is deleted but the bases in the flanking regions are mostly conserved. Such a pattern in the case of S. cerevisiae is shown in Figure 1. In mitochondria, the 3' leg is subjected to lot of variations which include changes as well as deletions and/or additions of bases. Thus the 3'leg of the 3' end fragment had been subjected to significant variations, probably due to selection pressures, in the course of evolution. Even though the AUG-context consensus sequence in mammalian mRNAs is not the same as that in yeast mRNAs, the sequence complementary to mammalian AUG-context consensus sequence is conserved in yeast 18S rRNA The need to test this conserved sequence for its functional ability in yeast has already been suggested by earlier workers (13). As can be seen in Figure 1, a part of anti-TP site has become part of anti-(AUG-context) sequence in the case of higher eukaryotes. Since such a motif in yeast corresponding to the anti-(AUG-context) sequence of mammals cannot base pair with AUG-context sequence of mRNAs from yeast, we thought that the anti-TP motif may still perform the same function of promoting translation-initiation as was proposed in the case of E. coli (15). In this paper, we report the results of our search on mRNAs from yeast for the TP site. Our analysis points out that the transcripts of only highly expressed genes inS. cerevisiae invariably possess such a TP site, which is free from any intramolecular secondary structure. The mRNA region between the TP site and the AUG codon folds into a weak hairpin stem to bring the TP site nearer to the AUG codon. The hairpin stem also brings distant nucleotides closer to the TP site so that the TP site extends its base pairing to an extended anti-TP site comprising the subsequences 5'-GGU-3' and the anti-TP site brought together by the central hairpin stem of the 3' end fragment of the ISS rRNA from yeast (Figure 1). The implications of such a site in relation to translationinitiation are discussed.
Method Based on the cluster analysis of relative synonymous codon usage, Sharp et al. (16) could identify two distinct groups of yeast genes. One group corresponds to the highly expressed genes that has extreme synonymous codon preference and the other group corresponds to lowly expressed genes. Other workers ( 17, 18) also have shown that relative rate of gene expression is correlated to the bias in the choice of synonymous codons in the coding region of yeast mRNAs. Hoekema et al. (4) could correlate the presence of preferred codons to gene expression by showing that replacement of an increasing number of preferred codons by non-preferred synonymous codons caused a dramatic decline of the expression level of PGK gene from yeast. The sequences of ribosomal RNAs and mRNAs used in this study were
1282
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taken from the EMBL nucleic acid sequence data base (Release 14, January 19SS) or from the list ofyeast genes as given byCigan and Donahue (13). The classification of genes from S. cerevisiae into two categories, such as highly expressed and lowly expressed, as given by Sharp eta/. (16) is used in our study. In the case of every gene, we considered the region comprising up to 60 nucleotides 5' to the AUG codon on mRNAs from these genes. These regions were searched for the occurrence of the TP site or a part thereof. The program COMPLEMENT (19) reported in our earlier work was used to check whether the sites thus picked up were free from intramolecular base pairing. Only those sites oflength > = 3 consecutive bases, that did not have any possible intramolecular base pairing were picked up as valid sites.
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Results and Discussion Conservation of the Anti-TP Site (5'-GGAUCA-3') in 18S rRNAs It is well known that in ISS rRNAs from eukaryotes, the pyrimidine-rich region corresponding to the anti-Shine-Dalgarno sequence is absent. Our examination of the small subunit rRNA sequences from 40 eukaryotic species as listed in an aligned form (20) reveals that all the bases of the anti-TP site are conserved without exception. The secondary structure derived for ISS rRNA ofS. cerevisiae (21) using comparative sequence analysis, shows that the bases of the anti-TP site are in a single-stranded form. Moreover, by using kethoxal probe Hogan eta/. (22) have shown that this site is accessible and single-stranded. The strong conservation, single-stranded nature, Table I List of the mRNAs of Lowly Expressed Genes From S. cerevisiae Gene (Identifier)"
TP Site
Gene (Identifier)"
TP Site
gall (SCGALS2) gall (SCGAL7) cbpl (SCCBPl) cbp2 [CBP2J trp5 (SCTRP5A) eilv2 (SCILV2) sir3 (SCSIR3) atp2 (SCATP21) rasl (SCRASHOl) cox6 (SCCOX6) ade4 (SCADE4) carl (SCCAR) hisl (SCHIOl) cpal (SCCPAl) rad2 (SCRAD2G) spt2 (SCSPT2) pho3 (SCPH035) gcn4 (SCGCN4)
None None None None None None None None None None None None None None None None None None None None
ga/4 (SCGAL4) ga/10 (SCGALS2) cbp6 (SCCBP6) trpl (SCTRPl) ilvl (SCILVl) sir2 (SCSIR2) arg4 (SCARG4) cupl (SCCUP 1) ura3 (SCURA3) matal (SCMATI) mata2 (SCMAT2) omp (SCOMPMil) his4 (SCHIS4) cpa2 (SCCPA2) rad7 (SCRAD7) spt3 (SCSPT3) pho5 (SCPH035) hstl [HSTIJ
None None None None None None None None None None None None None None None None None None None None
iso-2-cytochrome c (SCCYT2) Citrate synthetase (SCCSOl)
B-tubulin (SCBTUB) Invertase (SCINVE)
"Gene name is followed by the identifier. EMBL identifier is given in round brackets; identifier as used in Cigan and Donahue (13) is given in square brackets.
Translation-Initiation Promoting Site
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and accessibility of this site suggest that this is an important site which may be involved in molecular interaction.
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TP Site on mRNAs of Highly Expressed Genes From S. cerevisiae
We have analysed all the genes used by Sharp et al. (16) except those in which the sequence information about the 5' region was incomplete. The information on the transcription start site for the genes was taken from EMBL data base or from Cigan and Donahue (13). None of the genes from the lowly expressed class had the TP site. The list of 40 lowly expressed genes is given in Table I. The results of the search for TP site on 24 genes from the highly expressed class are given in Table II. As can be seen from the Table II, all the highly expressed genes except three, namely rpll6, rpll7A, and rp51A (numbered 4, 6 and 15), have got a subsequence (length>= 3) from the hexamer TP site. The three exceptions have subsequences AgCC, AcaC and GAgaC which can still form base pairing with anti-TP site by involving A· G and A · C base pairs. A · G and G · U base pairs have been observed to occur in rRNA structures (23). The bases A and C have the potential to form a protonated base pair with a geometry similar to that of the G · U base pair (24). The presence of the sites only on highly expressed genes indicates that there exists a correlation between the existence of this site on mRNA and the high rate of translation.
It can be seen from Table II that the locations of the TP site on mRNAs are varying. In thecaseofpgapll,hsp90andgdhl (numbered as 17, 18and 19), thesitesoccurbeyond50 nucleotides upstream of AUG codon. In all the remaining cases, they occur in a wide range in the region of 4 to 33 nucleotides upstream of AUG codon. Even though in the case ofE. coli, we observed a similar variation in the location ofTP site (15), it was acceptable because the TP site was presumed to act in concert with the SD site. However, in the case of yeast, there has not been any indication in the available literature, of cis-acting elements that would act via base pairing with rRNA In view of this, we decided to check whether the observed TP site on mRNAs from yeast could come closer to AUG codon by the formation of intra-mRNA secondary structure. We worked out the possible secondary structure of the region between TP site and AUG codon on the mRNAs from highly expressed genes. They are shown in Figure 2. In addition to regular Watson-Crick base pairs, the mismatch base pairs A· G, G · U, and A· C were allowed with the condition that in a hairpin stem, if mismatch base pairs occurred, they should always be accompanied by Watson-Crick base pairs. The hairpins thus formed comprise, in total, 102 base pairs out of which 80 are Watson-Crick type. Out of the 22 non-canonical base pairs, 8 are of the type A· C and II are of the type A· G. The high occurrence of A· C and A· Gover G · U base pairs is probably due to the overall richness of A residues in the leader regions of mRNAs from yeast (13). The analysis brought out the following features: i) It is found that in all those cases, where the TP site occurs at loctions > 12 nucleotides upstream of AUG, hairpin stems could be formed. The mRNAs numbered 2, 4, 5, 11,21-24, in which we could not find hairpin stems (Figure 2), have the TP sites at locations -9, -8,-12, -8, -7, -7,-7 and -6,respectively. Ifone accepts hairpin stems of only A · G base pairs, the mRNA numbered 5 will have a hairpin stem.
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Table II Results of the Search for the TP Site on mRNAs of Highly Expressed Genes From S. cerevisiae No.
Gene
(Identifier)"
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2 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.
adr2 rpslO rp/29 rpl16 rp/34 rpl17A rp/25 H3(II) rpsl6A
PGK rp28 rps31 H3(1)
EFloA rp51A
Actin pgapll hsp90 gdhl hxk2 rp/46 rps24 rp29 pen046
(SCADR2) (S10) (SCCYH2) (SCRIBL16) (SCRPL34) (SCRIBL17) (SCRIBL25) (SCH3H402) (Sl6A) (SCPGK5) (SCRP28A1) (SCRPS3l) [H3(1)) [EFlaA) (rp51A) (SCACTl) (SCGAP3) (SCHSP90) (SCGDHN) (SCHXK2) (SCRPL46) (SCRPS24) (SCRP29) (SCENOA)
TP site on mRNA Sequence UGAUCCb 3
Location
Journal of Biomolecular Structure and Dynamics Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tbsd20
Translation-Initiation Promoting Site on Transcripts of Highly Expressed Genes From Saccharomyces cerevisiae and the Role of Hairpin Stems to Position the Site Near the Initiation Codon a
T. A. Thanaraj & M. W. Pandit
a
a
Centre for Cellular and Molecular Biology , Hyderabad , 500 007 , India Published online: 21 May 2012.
To cite this article: T. A. Thanaraj & M. W. Pandit (1990) Translation-Initiation Promoting Site on Transcripts of Highly Expressed Genes From Saccharomyces cerevisiae and the Role of Hairpin Stems to Position the Site Near the Initiation Codon, Journal of Biomolecular Structure and Dynamics, 7:6, 1279-1289, DOI: 10.1080/07391102.1990.10508565 To link to this article: http://dx.doi.org/10.1080/07391102.1990.10508565
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.
Downloaded by [Rutgers University] at 22:23 08 April 2015
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 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, /SSN 0739-1102 Volume 7, Issue Number 6 (1990), "'Adenine Press (1990).
Translation-Initiation Promoting Site on Transcripts of Highly Expressed Genes From Saccharomyces cerevisiae and the Role of Hairpin Stems to Position the Site Near the Initiation Codon Downloaded by [Rutgers University] at 22:23 08 April 2015
T.A. Thanaraj and M.W. Pandit* Centre for Cellular and Molecular Biology Hyderabad 500 007, India Abstract It is reported that the AUG-upstream region on mRNAs of highly expressed genes from S. cerevisiae invariably possesses a translation-initiation promoting site, that can base pair with a well-conserved site on 18S rRNA during the formation of 40S initiation complex. Weak hairpin stem in the mRNA region between such a site and the initiation codon brings the site nearer to the initiation codon and also extends the length of base pairing. Such a base pairing can lead to a comparatively more stable 40S initiation complex, resulting in a higher rate of formation of the 80S initiation complex and consequently in an enhancement ofthe rate of initiation of translation. The site on 18S rRNA can interchange its base pairing between the site on mRNA and a well-conserved site on 25S rRNA in the formation of the 80S initiation complex.
Introduction The differential levels of the cellular proteins in eukaryotes can be attributed to the controls due to the various steps involved in the pathway from gene to protein, the major ones being mRNA transcription, processing, transport to the cytoplasm, and translation. The turnover rates of the mRNAs and proteins also play a role to this effect. There is much evidence to support the view that a significant degree of translational control occurs in eukaryotes (1,2). The controls for translational efficiency are exerted through processes such as initiation complex formation, chain elongation and termination. Even though the translational efficiency may be influenced by the level of elongation (3,4), the primary controlling factor is the rate of initiation (1,5-7). The scanning model for translation in eukaryotes as proposed and updated recently by Kozak (8) implies that the small ribosomal subunit binds initially at/near the 5' cap structure of the mRNA and then migrates along the mRNA to stop at the first AUG codon that is in a favourable context for initiating translation. Since every eukaryotic mRNA is *Author to whom correspondence should be addressed.
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Thanaraj & Pandit
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1280
capped, the difference in the rate of initiation must be governed by factors other than the cap structure. Secondary structure of mRNA in the 5' region has been shown to play a role (9-12). Consensus sequences in the AUG-context, which can act as signals for recognition of the functional AUG codon, have also been identified for both yeast and mammalian mRNAs (13,14). Mutations in the AUG-context sequence (5'-CC~CCAUGG-3') of mammalian mRNAs modulate the levels of translation-initiation to the extent of >20-fold (14). The mutations in the AUGcontext sequence (5'{AtAAUGU-3') of yeast mRNAs modulate the translationinitiation efficiency up to 2-fold (12), even though the extent of such a modulation is not as high as in the case of mammalian mRNAs. Cigan and Donahue (13) could identify differences in base composition of yeast leader regions- highly and lowly expressed genes having A- and G-richness, respectively. Leader-shuffiing experiments have documented the ability of certain cellular and viral 5' sequences to support efficient translation (8). Even though the AUG-context sequence in mammals has been proposed to base pair with a motif on 18S rRNA(l4), the role of mRNA-rRNA base pairing interactions in the scanning model have not been well characterised. The role of such a base pairing which may enhance the stability of the 40S initiation complex cannot be ruled out. Highly stable 40S initiation complex will lead to a higher rate of formation of 80S initiation complex leading to the enhanced translational yield. The involvement of the 3' end fragment (corresponding to the colicin fragment in E. coli) ofrRNA from the small subunit, in the initiation of translation is sufficiently well established in prokaryotes. The 3' end fragment of 16S rRNAfromE. coli contains the anti -Shine-Dalgamo sequence. Recently, we have reported sufficient suggestive evidence for the involvement of the hexamer (5'-GGAUCA-3'), from the abovementioned fragment, in the promotion ofthe translation-initiation by virtue ofbase pairing with a translation-initiation promoting (TP) site on mRNAs of highly expressed genes from E. coli (15). The 3' end fragment of the small subunit rRNA from E. coli as well as S. cerevisiae is shown in Figure 1. In mammalian cells, the involvement of the motif 5'-GGUGG-3' in base pairing with the AUG-context G A
G A G
c
G
A
G C A U U • G G c
g g A A
:
3'
I
A U U
U G G A
U G C C
c a ~s c D
u u aTP c
5'-ACAAggy _____ ggAUCA
~
S •- ACAAGGU
A C C U
G C G G
~ aTP G"iiifiCAUUA-3'
Extended a.TP
.S. cereuisiae Figure 1: Central hairpin stem of the 3' end fragment of the small subunit rRNAs. The site equivalent to the anti-(AUG-context) sequence of mammalian rRNA is indicated (dashed line). Watson-Crick base pairing is shown by colon(:); A· G and G · U by small circle. aTP, anti-TP site; aSD, anti-SD site.
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sequence has been suggested (14). Such a motif comprises of subsequences 5'-GGU-3' and 5'-GG-3' (shown by dashed line in Figure 1) held together by the central hairpin stem of the 3' end fragment of the 18S rRNA The 3'legofthe central hairpin stem is particularly interesting. In E. coli, as shown in Figure 1, this leg is formed by the sequence 5'-GGAUCACCUCCUUA-3', which consists of anti-SD (aSD) region flanked by an additional hexamer (designated as aTP) and the 3' terminal bases. In archaebacteria and chloroplast, this pattern as well as the individual bases therein are well conserved. In eukaryotes, the anti-SD region is deleted but the bases in the flanking regions are mostly conserved. Such a pattern in the case of S. cerevisiae is shown in Figure 1. In mitochondria, the 3' leg is subjected to lot of variations which include changes as well as deletions and/or additions of bases. Thus the 3'leg of the 3' end fragment had been subjected to significant variations, probably due to selection pressures, in the course of evolution. Even though the AUG-context consensus sequence in mammalian mRNAs is not the same as that in yeast mRNAs, the sequence complementary to mammalian AUG-context consensus sequence is conserved in yeast 18S rRNA The need to test this conserved sequence for its functional ability in yeast has already been suggested by earlier workers (13). As can be seen in Figure 1, a part of anti-TP site has become part of anti-(AUG-context) sequence in the case of higher eukaryotes. Since such a motif in yeast corresponding to the anti-(AUG-context) sequence of mammals cannot base pair with AUG-context sequence of mRNAs from yeast, we thought that the anti-TP motif may still perform the same function of promoting translation-initiation as was proposed in the case of E. coli (15). In this paper, we report the results of our search on mRNAs from yeast for the TP site. Our analysis points out that the transcripts of only highly expressed genes inS. cerevisiae invariably possess such a TP site, which is free from any intramolecular secondary structure. The mRNA region between the TP site and the AUG codon folds into a weak hairpin stem to bring the TP site nearer to the AUG codon. The hairpin stem also brings distant nucleotides closer to the TP site so that the TP site extends its base pairing to an extended anti-TP site comprising the subsequences 5'-GGU-3' and the anti-TP site brought together by the central hairpin stem of the 3' end fragment of the ISS rRNA from yeast (Figure 1). The implications of such a site in relation to translationinitiation are discussed.
Method Based on the cluster analysis of relative synonymous codon usage, Sharp et al. (16) could identify two distinct groups of yeast genes. One group corresponds to the highly expressed genes that has extreme synonymous codon preference and the other group corresponds to lowly expressed genes. Other workers ( 17, 18) also have shown that relative rate of gene expression is correlated to the bias in the choice of synonymous codons in the coding region of yeast mRNAs. Hoekema et al. (4) could correlate the presence of preferred codons to gene expression by showing that replacement of an increasing number of preferred codons by non-preferred synonymous codons caused a dramatic decline of the expression level of PGK gene from yeast. The sequences of ribosomal RNAs and mRNAs used in this study were
1282
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taken from the EMBL nucleic acid sequence data base (Release 14, January 19SS) or from the list ofyeast genes as given byCigan and Donahue (13). The classification of genes from S. cerevisiae into two categories, such as highly expressed and lowly expressed, as given by Sharp eta/. (16) is used in our study. In the case of every gene, we considered the region comprising up to 60 nucleotides 5' to the AUG codon on mRNAs from these genes. These regions were searched for the occurrence of the TP site or a part thereof. The program COMPLEMENT (19) reported in our earlier work was used to check whether the sites thus picked up were free from intramolecular base pairing. Only those sites oflength > = 3 consecutive bases, that did not have any possible intramolecular base pairing were picked up as valid sites.
Downloaded by [Rutgers University] at 22:23 08 April 2015
Results and Discussion Conservation of the Anti-TP Site (5'-GGAUCA-3') in 18S rRNAs It is well known that in ISS rRNAs from eukaryotes, the pyrimidine-rich region corresponding to the anti-Shine-Dalgarno sequence is absent. Our examination of the small subunit rRNA sequences from 40 eukaryotic species as listed in an aligned form (20) reveals that all the bases of the anti-TP site are conserved without exception. The secondary structure derived for ISS rRNA ofS. cerevisiae (21) using comparative sequence analysis, shows that the bases of the anti-TP site are in a single-stranded form. Moreover, by using kethoxal probe Hogan eta/. (22) have shown that this site is accessible and single-stranded. The strong conservation, single-stranded nature, Table I List of the mRNAs of Lowly Expressed Genes From S. cerevisiae Gene (Identifier)"
TP Site
Gene (Identifier)"
TP Site
gall (SCGALS2) gall (SCGAL7) cbpl (SCCBPl) cbp2 [CBP2J trp5 (SCTRP5A) eilv2 (SCILV2) sir3 (SCSIR3) atp2 (SCATP21) rasl (SCRASHOl) cox6 (SCCOX6) ade4 (SCADE4) carl (SCCAR) hisl (SCHIOl) cpal (SCCPAl) rad2 (SCRAD2G) spt2 (SCSPT2) pho3 (SCPH035) gcn4 (SCGCN4)
None None None None None None None None None None None None None None None None None None None None
ga/4 (SCGAL4) ga/10 (SCGALS2) cbp6 (SCCBP6) trpl (SCTRPl) ilvl (SCILVl) sir2 (SCSIR2) arg4 (SCARG4) cupl (SCCUP 1) ura3 (SCURA3) matal (SCMATI) mata2 (SCMAT2) omp (SCOMPMil) his4 (SCHIS4) cpa2 (SCCPA2) rad7 (SCRAD7) spt3 (SCSPT3) pho5 (SCPH035) hstl [HSTIJ
None None None None None None None None None None None None None None None None None None None None
iso-2-cytochrome c (SCCYT2) Citrate synthetase (SCCSOl)
B-tubulin (SCBTUB) Invertase (SCINVE)
"Gene name is followed by the identifier. EMBL identifier is given in round brackets; identifier as used in Cigan and Donahue (13) is given in square brackets.
Translation-Initiation Promoting Site
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and accessibility of this site suggest that this is an important site which may be involved in molecular interaction.
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TP Site on mRNAs of Highly Expressed Genes From S. cerevisiae
We have analysed all the genes used by Sharp et al. (16) except those in which the sequence information about the 5' region was incomplete. The information on the transcription start site for the genes was taken from EMBL data base or from Cigan and Donahue (13). None of the genes from the lowly expressed class had the TP site. The list of 40 lowly expressed genes is given in Table I. The results of the search for TP site on 24 genes from the highly expressed class are given in Table II. As can be seen from the Table II, all the highly expressed genes except three, namely rpll6, rpll7A, and rp51A (numbered 4, 6 and 15), have got a subsequence (length>= 3) from the hexamer TP site. The three exceptions have subsequences AgCC, AcaC and GAgaC which can still form base pairing with anti-TP site by involving A· G and A · C base pairs. A · G and G · U base pairs have been observed to occur in rRNA structures (23). The bases A and C have the potential to form a protonated base pair with a geometry similar to that of the G · U base pair (24). The presence of the sites only on highly expressed genes indicates that there exists a correlation between the existence of this site on mRNA and the high rate of translation.
It can be seen from Table II that the locations of the TP site on mRNAs are varying. In thecaseofpgapll,hsp90andgdhl (numbered as 17, 18and 19), thesitesoccurbeyond50 nucleotides upstream of AUG codon. In all the remaining cases, they occur in a wide range in the region of 4 to 33 nucleotides upstream of AUG codon. Even though in the case ofE. coli, we observed a similar variation in the location ofTP site (15), it was acceptable because the TP site was presumed to act in concert with the SD site. However, in the case of yeast, there has not been any indication in the available literature, of cis-acting elements that would act via base pairing with rRNA In view of this, we decided to check whether the observed TP site on mRNAs from yeast could come closer to AUG codon by the formation of intra-mRNA secondary structure. We worked out the possible secondary structure of the region between TP site and AUG codon on the mRNAs from highly expressed genes. They are shown in Figure 2. In addition to regular Watson-Crick base pairs, the mismatch base pairs A· G, G · U, and A· C were allowed with the condition that in a hairpin stem, if mismatch base pairs occurred, they should always be accompanied by Watson-Crick base pairs. The hairpins thus formed comprise, in total, 102 base pairs out of which 80 are Watson-Crick type. Out of the 22 non-canonical base pairs, 8 are of the type A· C and II are of the type A· G. The high occurrence of A· C and A· Gover G · U base pairs is probably due to the overall richness of A residues in the leader regions of mRNAs from yeast (13). The analysis brought out the following features: i) It is found that in all those cases, where the TP site occurs at loctions > 12 nucleotides upstream of AUG, hairpin stems could be formed. The mRNAs numbered 2, 4, 5, 11,21-24, in which we could not find hairpin stems (Figure 2), have the TP sites at locations -9, -8,-12, -8, -7, -7,-7 and -6,respectively. Ifone accepts hairpin stems of only A · G base pairs, the mRNA numbered 5 will have a hairpin stem.
1284
Thanaraj & Pandit
Table II Results of the Search for the TP Site on mRNAs of Highly Expressed Genes From S. cerevisiae No.
Gene
(Identifier)"
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2 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.
adr2 rpslO rp/29 rpl16 rp/34 rpl17A rp/25 H3(II) rpsl6A
PGK rp28 rps31 H3(1)
EFloA rp51A
Actin pgapll hsp90 gdhl hxk2 rp/46 rps24 rp29 pen046
(SCADR2) (S10) (SCCYH2) (SCRIBL16) (SCRPL34) (SCRIBL17) (SCRIBL25) (SCH3H402) (Sl6A) (SCPGK5) (SCRP28A1) (SCRPS3l) [H3(1)) [EFlaA) (rp51A) (SCACTl) (SCGAP3) (SCHSP90) (SCGDHN) (SCHXK2) (SCRPL46) (SCRPS24) (SCRP29) (SCENOA)
TP site on mRNA Sequence UGAUCCb 3
Location
