Institute of Cell and Molecular Biology, University of Edinburgh, King's Buildings, Mayfield Road, ... +Present address: MRC Laboratory of Molecular Biology, Hills Road, .... experiments the S7 protein has been demonstrated to interact with.
Q-DI 1995 Oxford University Press
4616-4619 Nucleic Acids Research, 1995, Vol. 23, No. 22
Cloning and characterisation of the gene encoding the ribosomal protein S5 (also known as rpl4, Saccharomyces cerevisiae
S2,
YS8) of
Olga lgnatovich1'+, Michelle Cooper, Helen M. Kulesza and Jean D. Beggs* Institute of Cell and Molecular Biology, University of Edinburgh, King's Buildings, Mayfield Road, Edinburgh EH9 3JR, UK EMBL accession no. X89368
Received August 18, 1995; Revised and Accepted October 16, 1995
ABSTRACT The protein sequence derived from a cloned yeast gene and partial cDNA has high sequence identity to 40S ribosomal subunit S5 proteins of higher eukaryotic origin. The open reading frame of the gene is flanked by consensus sequence motifs characteristic of ribosomal protein genes and the pattem of transcription of the gene in yeast cells subjected to nutritional shift or temperature shock is also typical of a ribosomal protein gene. The gene is single copy and essential for viability. The predicted sequence of the N-terminus of the protein identifies it as a phosphorylated ribosomal protein variously known as rpl 4, S2 or YS8, the least basic of the non-acidic ribosomal proteins of Saccharomyces cerevisiae.
INTRODUCTION The ribosomal proteins from all eukaryotes have been highly conserved through evolution and sequence similarities among yeast and rat ribosomal proteins indicate that there is likely to be a one-to-one correspondence among ribosomal proteins of eukaryotes from yeast to mammals (1-3). Ribosome content per cell varies under different physiological conditions in proportion to the cell growth rate and the production of ribosomal proteins and rRNAs increases or decreases according to requirement. Ribosomal protein synthesis is subject to coordinate regulation to ensure the production of equivalent amounts of each ribosomal component. In yeast the ribosomal proteins are balanced primarily through regulation of transcription of their genes (1,4). Many ribosomal proteins are posttranslationally modified by phosphorylation, methylation or acetylation, but the significance of the modifications remains unknown. Here we describe the cloning and characterisation of the gene encoding ribosomal protein S5 (previously designated S2, rpl4 or YS8) of Saccharomyces cerevisiae which corresponds to the S5 protein of higher eukaryotic ribosomes and S7 of prokaryotes and plastids. This is one of the few phosphorylated ribosomal proteins in S.cerevisiae. *
MATERIALS AND METHODS Strains and microbiological procedures S.cerevisiae strain W303 [MATalk ade2-1/ade2-1 canl- 100/canl100 trpl-1/trpl-J leu2-3,112/leu2-3,112 his3-11,15/his3-11,15 ura3/ura3; (5)] was used for the gene disruption, copy number and expression studies. Escherichia coli DH5a F' (6) was used for general propagation of plasmid DNAs. E.coli NM522 (7) was used to propagate the yeast genomic library [in YEp13;(8)]. Yeast transformation was by the lithium acetate procedure (9). Nucleic acid methods 1400 colonies of E.coli strain NM522 transformed with the S.cerevisiae genomic library (8) were screened by colony hybridisation (10), probing with unifonmly 32P-labelled (11) 712 bp cDNA. Three plasmids were isolated that contained overlapping fragments. DNA fragments were cloned in pBluescript KS+/- and single stranded DNA sequencing was carried out by the dideoxy chain termination method using Sequenase Version 2.0 (USB). The DNA sequence (EMBL accession no. X89368) was determined on both strands. The cDNA::HIS3 disruption constructs were made by inserting the HIS3 gene on a 1.8 kb BamHI fragment [from YIpl (12)] at the unique BclI site in the cDNA or by replacing the 89 basepairs between the BclI and HpaI sites with a 1.3kb XhoLlBamHI fragment (XhoI end filled-in) carrying the HIS3 gene. In each case the direction of transcription of HIS3 was opposite to that of the disrupted sequence. For Southern blots, yeast genomic DNA was isolated (13), digested with restriction endonucleases, fractionated by electrophoresis in 0.8% (w/v) agarose, blotted to Hybond-N membrane (Amersham) and probed with the cDNA or the HIS3 gene (to confirm the gene disruption). Computer analysis of the DNA sequence was performed using the BLASTN, BLASTP (14) and PILEUP (15) programs implemented on the National Center for Biotechnology Information (USA) and the European Bioinformatics Institute facilities (Cambridge, UK) respectively.
To whom correspondence should be addressed
+Present address: MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK
Nucleic Acids Research, 1995, Vol. 23, No. 22 4617
RESULTS AND DISCUSSION Isolation and characterisation of the genomic sequence The yeast RPS5 sequence was initially identified as a 720 bp partial cDNA selected from a kgtl 1 library through its cross-reaction with a human Sm auto-antiserum (H.K., M. Dalrymple and J.D.B. to be published elsewhere). The cloned cDNA was used as a probe to screen a yeast genomic library (8) and the complete gene and flanking sequences were cloned. The genomic sequence (EMBL accession no. X89368) contains one long open reading frame encoding a predicted polypeptide of 225 amino acids. No intron canonical sequences were identified either within or flanking the open reading frame and the sequence of the partial cDNA clone was identical from the EcoRI site at nucleotide +55 (with respect to the putative initiating methionine codon) to nucleotide +770 of the genomic sequence, indicating 95 bases of 3' untranslated sequence in the mRNA. The initiation codon occurs in the context 5'-CAAAGATGTCT-3' which differs by only one nucleotide from the general consensus 5'-AIYAA/TAAATGTCT-3' characteristic for genes of S.cerevisiae (16) and is preceded by in-frame nonsense codons at positions -24 and -18. Possible TATA sequences (TAATTA and TAATA) are present at -60 and -43. AATAA at -20 may represent a transcription start site (17,18). Sequences that are typically found upstream of ribosomal protein genes (RPG) are also present. Two sequences, ACACCCATATACCC (-479) and TAACCCATACATAT (-454), are good candidates for UASRpG elements [consensus ACACCCATACATTr' (19)] which are transcriptional control elements and binding sites for the transcription factor RAPI (reviewed in ref. 1). Downstream of the UASRpG elements a T-rich sequence normally functions as a constitutive promoter element and several candidate sequences occur here.
Gene copy number, chromosome assignment and disruption Many proteins of the yeast translational apparatus, including the majority of the yeast ribosomal proteins, are encoded by two genes (4). To investigate the copy number of this gene, yeast genomic DNA was digested with EcoRP, Sall, EcoRI plus Sall or HindIll and analysed by Southern blotting, using the cloned cDNA as probe. In each case only a single band was detected, indicating that this is a single copy gene in S.cerevisiae (data not shown). Probing of an OFAGE blot indicated that the gene is located on chromosome X (data not shown). This was subsequently confirmed when the complete sequence of chromosome X was released (ORF:YJR123w; Rose,M., Koetter,P. and Entian,K.D., unpublished results). To determine whether this sequence is essential for yeast viability, two cDNA::HIS3 disruption constructs were made. In one construct a fragment containing the yeast HIS3 gene was inserted into the unique Bcll site, while in the other the region between the HpaI and Bcll sites was replaced by the HIS3 DNA fragment. The cDNA::HIS3 constructs were transformed into the his3 diploid strain W303 and the anticipated disruption of one chromosomal locus was confirmed by Southern blot analysis (not shown). Dissection of a total of 13 tetrads from the disrupted diploids (nine having the HIS3 gene inserted at the Bcll site, four with the deletion) resulted in the recovery of only two viable spores in each case and all were his-. Therefore this gene encodes an essential function.
A
Time after shift ethanol to glucose
CC;
0
5
10 30 60 CE
3
4
S It:.
1
2
B
5
6
7
Time at 37"C
0 10 30 60
123 l
2
3
4
Figure 1. Northern analysis of the effects on RPS5 transcript of (A) carbon source upshift and (B) heat-shock. (A) For the nutritional shift, a culture was grown at 30°C to mid-log phase in minimal medium containing 2% (v/v) ethanol as sole carbon source and then glucose was added to 2% (v/v). Lanes 2-6, RNA extracted from cells 0, 5, 10, 30 or 60 min after the introduction of glucose to the growth medium. Lanes 1 and 7 are controls, showing RNA from cells grown continuously on glucose (CG) or cells prepared as for the carbon source upshift but maintained in ethanol as sole carbon source for a further 60 min (CE). (B) Yeast cultures were subjected to heat-shock by growth in YPDA at 23°C to mid-log phase and addition of an equal volume of medium at 49°C, followed by incubation at 37°C for the times indicated. For Northern blots, yeast total RNA was extracted (21) and analysed by electrophoresis in 1.5% (w/v) agarose/formaldehyde gels (22), blotted to Hybond-N membrane and probed with the RPS5 cDNA. Equivalent loading of different RNA samples was checked by ethidium bromide staining of the rRNA and tRNA.
Expression studies To provide further evidence that the yeast RPS5-related gene encodes a ribosomal protein, the pattern of transcription was investigated upon subjecting yeast cells to nutritional and temperature shifts. Carbon source upshift of yeast cells from glycerol to glucose medium results in a 2- or 3-fold increase in the rate of transcription of ribosomal protein genes (20). Yeast strain W303 was grown to logarithmic phase on ethanol as sole carbon source and then glucose was added to the growth medium. Within 5 min of the carbon source change the level of the RPS5-related transcript had increased and continued to increase during 60 min incubation in glucose compared to the starting level (Fig. 1 A, compare lane 2 with lanes 3-6) and compared to a control culture to which no glucose was added (Fig. IA, lane 7). Yeast cells subjected to heat-shock by shifting abruptly from 23 to 370C undergo a complex pattern of changes; production of some proteins is repressed while production of others is induced. The synthesis of ribosomal proteins is particularly sensitive to heat-shock, dropping by -80% within 20 min. This effect results
4618 Nucleic Acids Research, 1995, Vol. 23, No. 22
from a transient decrease in the rate of transcription of ribosomal protein genes and by 60 min after the temperature shift, ribosomal protein synthesis is generally restored to normal. Ten minutes after submitting a logarithmic yeast culture to heat-shock the level of RPS5-related transcript was dramatically reduced but, as expected for a typical ribosomal protein mRNA, the effect was highly transient (Fig. 1 B). Thus with respect to nutritional as well as temperature shifts, transcription of this gene is typical of that for a yeast ribosomal protein gene.
Protein sequence comparisons The open reading frame of the cloned gene predicts a protein product with a calculated molecular weight of 25 038 kDa and pl of 9.11. Omitting the initial methionine, the N-terminal amino acid sequence derived from the genomic sequence matches the N-terminal sequence [in 19 of the 20 residues reported (23)] of the ribosomal protein rpl4 [also known as S2 or YS8 (4)] a component of the 40S subunit of S.cerevisiae (23). This is the least basic of the non-acidic ribosomal proteins of S.cerevisiae and it is phosphorylated (24). In a computer database search the predicted protein sequence had the highest identities (Table 1) to the ribosomal protein S5 from human (Frigerio, J.M., Dagom, J.C. and lovanna, J.L., unpublished data), rat (25) and the hydrozoan Podocryne carnea (26). These matches indicate that this S.cerevisiae ribosomal protein is the homologue of the ribosomal protein S5 of higher eukaryotes. The gene has therefore been designated RPS5. Table 1. Amino acid identities between rpS5 of S.cerevisiae and ribosomal protein S5 of eukaryotes and S7 of prokaryotic or plastid origin
Origin
Accession no.
Amino acid identity (%)
Length/Gaps
Homo sapiens
U 14970
70.9
187/2
Rattus rattus
P24050
70.4
187/2
Podocryne carnea
Q08364
68.8
187/2
Desulfurococcus mobilis Bacillus stearothermophilus
X73582
50.0
160/0
JG0008
34.4
122/5
Nicotiana tabacum
P06361
31.8
148/5
Zea mays chloroplast
M 17841
28.9
149/5
Escherichia coli
P02359
28.7
122/5
chloroplast
Only the carboxy-terminal residues show identity and the length of the aligned region is given in each case.
Significant identities with other ribosomal proteins include the small subunit ribosomal protein S7 of prokaryotic or chloroplast origin (Table 1). Amino acid sequence alignments reveal that the N-terminal end is variable in both sequence and length, whereas the C-terminus is highly conserved. As previously noted (25), the most carboxy-terminal 16 amino acids are particularly highly conserved in organisms spanning several kingdoms, implying that this region of the S5 and S7 proteins serves an important function.
The high degree of sequence similarity of the S5 and S7 proteins in such evolutionarily diverse organisms also suggests conservation of function(s) of this protein in the ribosome. Although very little is known about the eukaryotic protein, the S7 protein of Ecoli has been extensively studied. In cross-linking experiments the S7 protein has been demonstrated to interact with the 3' end of 16S rRNA (27-31), being crucial for the assembly of the 30S subunit (32). It contacts residues in the anticodon stem-loop of ribosome-bound tRNAs (33-35) and is the primary mRNA-binding protein in ribosomes (36). These interactions and ribosome structural studies (27) locate the S7 protein close to the decoding site of the ribosome. It will therefore be very interesting to determine whether these interactions are conserved in the eukaryotic protein and whether phosphorylation plays a role in regulating its function(s).
ACKNOWLEDGEMENTS We are extremely grateful to Jon Warner for very helpful suggestions and to Euan Gordon, ICMB, Edinburgh, for provision of the OFAGE blot. We thank The Darwin Trust of Edinburgh for support for 0.1. The Medical Research Council provided support for M.C. and H.M.K through a Studentship and a grant respectively. J.D.B. holds a Royal Society Cephalosporin Fund Senior Research Fellowship.
REFERENCES I Woolford, J.L. (1991) Adv. Genet., 29, 63-118. 2 Suzuki, K. and Wool, I. (1993) J. Biol. Chem., 268, 2755-2761. 3 Raue, H.A., Mager, W.H. and Planta, R.J. (1991) Structural and functional analysis of yeast ribosomal proteins. In Guthrie, C. and Fink, G.R. (eds), Guide to Yeast Genetics and Molecular Biology. Academic Press, New York, pp. 453-477. 4 Woolford, J.L. and Warner, J.R. (1991) The ribosome and its synthesis. In Broach, J.R., Pringle, J.R. and Jones, E.W. (eds), The Molecular and Cellular Biology of the Yeast SaccharomnYces cere0isiae Vol. 1. CSHL Press, New York, pp. 587-626. 5 Nasmyth, K. (1985) Cell, 42, 213-223. 6 Hanahan, D. (1983) J. Mol. Biol., 166, 557-580. 7 Gough, J.A. and Murray, N.E. (1983) J. Mol. Biol., 166, 1-19. 8 Nasmyth, N.A. and Tatchell, K. (1980) Cell, 19, 753-764. 9 Gietz, D., St.Jean, A., Woods, R. and Schiesti, R. (1992) Nucleic Acids Res., 20, 1425. 10 Grunstein, M. and Hogness, D.S. (1975) Proc. Natl. Acad. Sci. USA, 72, 3961-3965. 11 Feinberg, A.P. and Vogelstein, B. (1984) Anal. Biochem., 137, 266-267. 12 Struhl, K., Stinchcomb, D.T., Scherer, S. and Davis, R.W. (1979) Proc. Natl. Acad. Sci. USA, 76, 1035-1039. 13 Rose, M.D., Winston, F. and Hieter, P. (1990) Methods in Yeast Genetics. A Laboratory Course Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY 14 Altschul, S.F., Gish, W., Miller, W., Mye, E.W. and Lipman, D.J. (1990) J. Mol. Biol., 215, 403-410. 15 Devereux, J., Haeberli, P. and Smithies, 0. (1984) Nucleic Acids Res., 12, 387-395. 16 Cigan, A. and Donahue, T. (1987) Gene, 59, 1-18. 17 Hahn, S., Hoar, E. and Guarente, L. (1985) Proc. Natl. Acad. Sci. USA, 82, 8562-8566. 18 Guarente, L. (1987) Annu. Rev. Genet., 21, 425-452. 19 Planta, R.J. and Raue, H.A. (1988) Trends Genet., 4, 64-68. 20 Herruer, M.H., Mager, W.H., Woudt, L.P., Nieuwint, R.T.M., Wassernaar, G.M., Groeneveld, P. and Planta, R.J. (1987) Nucleic Acids Res., 15, 10 133-10 144. 21 Hopper, A.K., Banks, F. and Evangelidis, V. (1978) Cell, 14, 211-219. 22 Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Nucleic Acids Research, 1995, Vol. 23, No. 22 4619 23 Takakura, H., Tsunasawa, S., Miyagi, M. and Warner, J.R. (1992) J. Bio. Chem., 267, 5442-5445. 24 Kruse, C., Johnson, S.P. and Warner, J.R. (1985) Proc. Natl. Acad. Sci. USA, 82, 7515-7519. 25 Kuwano, Y., Olvera, J. and Wool, I.G. (1992) J. Biol. Chem., 267, 25 304-25 308. 26 Aerne, B.L., Baarder, C.D., Schmid, V. and Schuchert, P. (1993) Nucleic Acids Res., 21, 3575. 27 Brimacombe, R., Atmadja, J., Steige, W. and Schuler, D. (1988) J. Mol. Biol., 199,115-136. 28 Powers, T., Changchien, L.M., Craven, G.R. and Noller, H.F. (1988) J. Mol. Biol., 200, 309-319. 29 Dragon, F. and Brakier-Gingras, L. (1993) Nucleic Acids Res., 21, 1199-1203.
30 Dragon, F., Payant, C. and Brakier-Gingras, L. (1994) J. Mol. Biol., 244, 74-85. 31 Powers, T. and Noller, H.F. (1995) RNA, 1, 194-209. 32 Nowotny, V. and Nierhaus, K.H. (1988) Biochemistry, 27, 7051-7055. 33 Podkowinski, J. and Gomicki, P. (1989) Nucleic Acids Res., 17, 8767-8782. 34 Sylvers, L.A., Kopylov, A.M., Wower, J., Hixson, S.S. and Zimmermann, R.A. (1992) Biochemie, 74, 381-389. 35 Doring, T., Mitchell, P., Osswald, M., Bochkariov, D. and Brimacombe, R. (1994) EMBO J., 13, 2677-2685. 36 Stade, K., Rinke-Appel, J. and Brimacombe, R. (1989) Nucleic Acids Res., 17, 9889-9908.
