The EMBO Journal vol.9 no.4 pp. 1 253 - 1258, 1990
Temperature sensitive synthesis of transfer RNAs in vivo in Saccharomyces cerevisiae
R.Marschalek, D.Kalpaxis1 and Th.Dingermann Institut fir Biochemie der Medizinischen Fakultat, Universitiit Erlangen-Nurmberg, Fahrstrasse 17, D-8520 Erlangen, FRG 'Present address: Department of Biochemistry, School of Medicine, University of Patras, G-261 10 Patras, Greece
Communicated by H.-J.Gross
Dictyostelium discoideum tRNA genes can be expressed efficiently in vivo in yeast, and transcription products are processed to mature tRNAs. However, primnary transcripts of a variant tRNAVII(UAC) gene are processing deficient under standard growth conditions (30°C), due to a slightly altered 5' flanking region. A stable extended amino acid acceptor stem, which seems to be required to compensate a G5- G68 mismatch, cannot form. This mismatch destabilizes secondary and probably tertiary structures to such an extent that recognition of processing enzyme(s) under normal conditions (30°C) is impaired. Growing yeast cells at reduced temperature (22°C) can phenotypically complement the processing defect. This observation provides a new concept for the temperature dependent expression of protein coding genes which carry a nonsense codon. Translation of corresponding messages can be controlled by products of a temperature sensitive su-tRNA gene. We successfully tested this concept with two amber suppressors derived from a tRNAGIU(UUC) gene from D.discoideum. One of the variant tRNA genes codes for a product with a destabilized amino acid acceptor stem. Primary transcripts of this particular su-tRNAGlu(CUA) gene are processed only at reduced growth temperatures and consequently function as temperature sensitive suppressors only under these conditions. Key words: suppression/temperature sensitivity/tRNA processing/yeast
Introduction The synthesis of eukaryotic tRNA genes is controlled by gene internal promoter elements [for reviews see Sharp et al. (1985) and Geiduschek and Tocchini-Valentini (1988)]. In addition, a modulatory influence of 5' flanking regions has frequently been observed (Sprague et al., 1980; DeFranco et al., 1981; Dingermann et al., 1982; Hipskind and Clarkson, 1983; Shaw and Olson, 1984; Allison and Hall, 1985; Raymond et al., 1985; Loftquist and Sharp, 1986; Arnold and Gross, 1987; Dingermann et al., 1987). Regulation of tRNA gene transcription, however, has only been demonstrated in just a few cases (Sprague et al., 1977; Kuchino et al., 1987; Dingermann et al., 1988a). Therefore it is not surprising that systems which allow controlled expression of eukaryotic tRNA genes are not available with one exception: this system is based on gene amplification, which allows conditional expression of high levels of an Oxford University Press
amber suppressor tRNAser in monkey kidney cells (Sedivy et al., 1987). The tRNA gene is linked to the SV40 origin of replication. If a second DNA carrying a temperature sensitive SV40 large T antigen is cotransfected with the tRNA gene containing plasmid, the tRNA gene is highly amplified at the permissive temperature and consequently the corresponding tRNAser accumulates in the cell. During a recent study (Marschalek and Dingermann, 1988), where we analysed the influence of slightly altered 5' flanking regions on the efficiency of tRNA gene expression in vivo in yeast, an alternative approach towards deliberate regulation of tRNA synthesis became apparent. This approach is based on temperature sensitive maturation of the primary transcripts. Eukaryotic tRNA genes are transcribed yielding precursors which are consecutively processed to mature tRNA molecules. Maturation reactions include the removal of transcribed parts of the 5' and 3' flanking regions and the subsequent addition of the 3'-CCA terminus (Altman, 1978; Mazzara and McClain, 1980; Deutscher, 1984). Some primary transcripts contain introns which need to be removed (Abelson, 1979; Mao et al., 1980; Melton et al., 1980) and many nucleotides are methylated at distinct positions or hypermodified to sometimes very complex structures (for review see Bjork et al., 1987). The transcribed 5' and 3' flanking nucleotides appear to be removed by specific endonucleases (Garber and Altman, 1979; Garber and Gage, 1979; Hagenbuchle et al., 1979; Castano et al., 1985; Frendeway et al., 1985) and while in most studied cases removal of the 5' flanking region precedes removal of the 3' flanking region (Frendeway et al., 1985) alternative pathways have been discovered as well (Thomann et al., 1989). From many studies it became apparent that the recognition of enzymes which remove 5' leader and 3' trailer regions depends on an intact secondary and tertiary structure of the mature domain. This also applies to enzymes which are involved in intron splicing (Nishikura et al., 1982; Baldi et al., 1983; Mattoccia et al., 1983; Traboni et al., 1984; Willis et al., 1986; Nichols et al., 1988). In this report we describe a tRNA gene whose transcript naturally contains such a labile amino acid acceptor stem that transcribed flanking regions are needed in order to compensate this instability by forming a stable 'extended' amino acid acceptor stem. Transcripts of a gene variant carrying a deletion of nucleotides -2 and -3 within the 5' flanking region are processing deficient in vivo in yeast if cells are grown at 30°C. Processing does occur, however, in cells grown at 20 or 22°C. This concept of temperature sensitive synthesis of a tRNA was further tested by altering the amino acid acceptor stem of a tRNAGIu gene whose products are able to suppress amber mutations in yeast. Primary transcripts of the altered gene [su-tRNAGIU(CUA)3T] are processed only at or below 30°C, whereas cells grown at 37°C do not contain an active suppressor tRNA.
1253
R.Marschalek, D.Kalpaxis and Th.Dingermann
Results tRNAVaI(UAC) gene derivatives from Dictyostelium discoideum with wild-type mature tRNA coding region but aftered 5' flanking region produce processing deficient primary transcripts A tRNAVal(UAC) gene from D.discoideum has been shown to serve as efficient template in Saccharomyces cerevisiae (Dingermann et al., 1987; Marschalek and Dingermann, 1988). Primary transcripts made in vivo also undergo accurate 5' maturation as has been determined by primer extension analysis using an end labelled tRNAval(UAC) specific oligonucleotide (Dingermann et al., 1987; Marschalek and Dingermann, 1988). This primer is incubated with bulk tRNA isolated from yeast transformants and specifically anneals to nucleotides 24-40 of tRNAval(UAC) transcripts. A cDNA is synthesized with AMV reverse transcriptase and dNTPs, and products are separated on sequencing gels (Sanger et al., 1977). This has the advantage that not only transcription products of the tRNA gene can be detected specifically and with high sensitivity but that the 5' ends of the transcripts can also be identified. As shown in Figure 1, left lane, the tRNAVal (UAC) gene (wt) is expressed in vivo in yeast yielding primary transcripts which start at nucleotides -12 and -9. These primary transcripts are processed to tRNAs with mature 5' ends. Similarly, a variant of the tRNAval(UAC) gene, NF:8B, is expressed and processed in vivo in yeast, although with reduced efficiency (Figure 1, central lane). This drop in efficiency results from binding of a nuclear DNA binding protein which specifically recognizes the BamHI linker inserted at position -3 within the 5' flanking region of the tRNAval(UAC) region (Marschalek and
Dingermann, 1988). A remarkably different expression behaviour shows another tRNAval(UAC) variant gene, AN:8B. Although transcribed efficiently in vivo in yeast, primary transcripts accumulate and are not processed to mature size tRNAs (Figure 1, right lane). Plasmid AN:8B was fortuitously obtained during the cloning procedure of NF:8B and differs from NF:8B by a deletion of nucleotides -11, -3 and -2 in the wild-type 5' flanking region. Neither NF:8B nor AN:8B contains any alterations within the mature tRNA coding region or within the 3' flanking region with respect to the wild-type gene. The tRNAV8I(UAC) from D.discoideum contains an unusually labile amino acid acceptor stem It has been demonstrated that a highly ordered structure of primary transcripts and not any consensus sequence is responsible for recognition of tRNA processing enzymes (Garber and Altman, 1979; Nishikura et al., 1982, Baldi et al., 1983; Mattoccia et al., 1983; Traboni et al., 1984; Castano et al., 1985). The tRNAval(UAC) gene from D.discoideum codes for transcripts with a particularly unstable amino acid acceptor stem (see Figure 2a - c). In
addition to a G2 -U71 base pair there is also a G5 -G68 mismatch within this amino acid acceptor stem. Nevertheless, primary transcripts of the primary wild-type gene are processed correctly suggesting that the G5 -G68 mismatch plus the G2 -U71 base pair do not interfere with recognition requirements of processing enzymes. The processing deficiency of primary transcripts derived from AN:8B, however, points to the possibility that flanking regions might
1254
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Fig. 1. Primer extension analysis of tRNAs synthesized in vivo in yeast from a D.discoideum tRNAval(UAC) gene (wt) and from derivatives NF:8B and AN:8B. Bulk tRNA was isolated from yeast strains which were transformed with plasmids carrying the indicated tRNA genes. A 32P-end-labelled oligonucleotide recognizing exclusively products of the tRNAVa (UAC) gene and of its derivatives was hybridized with 0.25 A260 units of the respective bulk tRNAs. cDNAs were synthesized and size-fractionated on sequencing gels. An extended primer fragment of 40 nucleotides corresponds to mature tRNA (indicated by the small box). Larger cDNAs were synthesized on primary transcripts while fragments
Temperature sensitive synthesis of transfer RNAs in vivo in Saccharomyces cerevisiae
R.Marschalek, D.Kalpaxis1 and Th.Dingermann Institut fir Biochemie der Medizinischen Fakultat, Universitiit Erlangen-Nurmberg, Fahrstrasse 17, D-8520 Erlangen, FRG 'Present address: Department of Biochemistry, School of Medicine, University of Patras, G-261 10 Patras, Greece
Communicated by H.-J.Gross
Dictyostelium discoideum tRNA genes can be expressed efficiently in vivo in yeast, and transcription products are processed to mature tRNAs. However, primnary transcripts of a variant tRNAVII(UAC) gene are processing deficient under standard growth conditions (30°C), due to a slightly altered 5' flanking region. A stable extended amino acid acceptor stem, which seems to be required to compensate a G5- G68 mismatch, cannot form. This mismatch destabilizes secondary and probably tertiary structures to such an extent that recognition of processing enzyme(s) under normal conditions (30°C) is impaired. Growing yeast cells at reduced temperature (22°C) can phenotypically complement the processing defect. This observation provides a new concept for the temperature dependent expression of protein coding genes which carry a nonsense codon. Translation of corresponding messages can be controlled by products of a temperature sensitive su-tRNA gene. We successfully tested this concept with two amber suppressors derived from a tRNAGIU(UUC) gene from D.discoideum. One of the variant tRNA genes codes for a product with a destabilized amino acid acceptor stem. Primary transcripts of this particular su-tRNAGlu(CUA) gene are processed only at reduced growth temperatures and consequently function as temperature sensitive suppressors only under these conditions. Key words: suppression/temperature sensitivity/tRNA processing/yeast
Introduction The synthesis of eukaryotic tRNA genes is controlled by gene internal promoter elements [for reviews see Sharp et al. (1985) and Geiduschek and Tocchini-Valentini (1988)]. In addition, a modulatory influence of 5' flanking regions has frequently been observed (Sprague et al., 1980; DeFranco et al., 1981; Dingermann et al., 1982; Hipskind and Clarkson, 1983; Shaw and Olson, 1984; Allison and Hall, 1985; Raymond et al., 1985; Loftquist and Sharp, 1986; Arnold and Gross, 1987; Dingermann et al., 1987). Regulation of tRNA gene transcription, however, has only been demonstrated in just a few cases (Sprague et al., 1977; Kuchino et al., 1987; Dingermann et al., 1988a). Therefore it is not surprising that systems which allow controlled expression of eukaryotic tRNA genes are not available with one exception: this system is based on gene amplification, which allows conditional expression of high levels of an Oxford University Press
amber suppressor tRNAser in monkey kidney cells (Sedivy et al., 1987). The tRNA gene is linked to the SV40 origin of replication. If a second DNA carrying a temperature sensitive SV40 large T antigen is cotransfected with the tRNA gene containing plasmid, the tRNA gene is highly amplified at the permissive temperature and consequently the corresponding tRNAser accumulates in the cell. During a recent study (Marschalek and Dingermann, 1988), where we analysed the influence of slightly altered 5' flanking regions on the efficiency of tRNA gene expression in vivo in yeast, an alternative approach towards deliberate regulation of tRNA synthesis became apparent. This approach is based on temperature sensitive maturation of the primary transcripts. Eukaryotic tRNA genes are transcribed yielding precursors which are consecutively processed to mature tRNA molecules. Maturation reactions include the removal of transcribed parts of the 5' and 3' flanking regions and the subsequent addition of the 3'-CCA terminus (Altman, 1978; Mazzara and McClain, 1980; Deutscher, 1984). Some primary transcripts contain introns which need to be removed (Abelson, 1979; Mao et al., 1980; Melton et al., 1980) and many nucleotides are methylated at distinct positions or hypermodified to sometimes very complex structures (for review see Bjork et al., 1987). The transcribed 5' and 3' flanking nucleotides appear to be removed by specific endonucleases (Garber and Altman, 1979; Garber and Gage, 1979; Hagenbuchle et al., 1979; Castano et al., 1985; Frendeway et al., 1985) and while in most studied cases removal of the 5' flanking region precedes removal of the 3' flanking region (Frendeway et al., 1985) alternative pathways have been discovered as well (Thomann et al., 1989). From many studies it became apparent that the recognition of enzymes which remove 5' leader and 3' trailer regions depends on an intact secondary and tertiary structure of the mature domain. This also applies to enzymes which are involved in intron splicing (Nishikura et al., 1982; Baldi et al., 1983; Mattoccia et al., 1983; Traboni et al., 1984; Willis et al., 1986; Nichols et al., 1988). In this report we describe a tRNA gene whose transcript naturally contains such a labile amino acid acceptor stem that transcribed flanking regions are needed in order to compensate this instability by forming a stable 'extended' amino acid acceptor stem. Transcripts of a gene variant carrying a deletion of nucleotides -2 and -3 within the 5' flanking region are processing deficient in vivo in yeast if cells are grown at 30°C. Processing does occur, however, in cells grown at 20 or 22°C. This concept of temperature sensitive synthesis of a tRNA was further tested by altering the amino acid acceptor stem of a tRNAGIu gene whose products are able to suppress amber mutations in yeast. Primary transcripts of the altered gene [su-tRNAGIU(CUA)3T] are processed only at or below 30°C, whereas cells grown at 37°C do not contain an active suppressor tRNA.
1253
R.Marschalek, D.Kalpaxis and Th.Dingermann
Results tRNAVaI(UAC) gene derivatives from Dictyostelium discoideum with wild-type mature tRNA coding region but aftered 5' flanking region produce processing deficient primary transcripts A tRNAVal(UAC) gene from D.discoideum has been shown to serve as efficient template in Saccharomyces cerevisiae (Dingermann et al., 1987; Marschalek and Dingermann, 1988). Primary transcripts made in vivo also undergo accurate 5' maturation as has been determined by primer extension analysis using an end labelled tRNAval(UAC) specific oligonucleotide (Dingermann et al., 1987; Marschalek and Dingermann, 1988). This primer is incubated with bulk tRNA isolated from yeast transformants and specifically anneals to nucleotides 24-40 of tRNAval(UAC) transcripts. A cDNA is synthesized with AMV reverse transcriptase and dNTPs, and products are separated on sequencing gels (Sanger et al., 1977). This has the advantage that not only transcription products of the tRNA gene can be detected specifically and with high sensitivity but that the 5' ends of the transcripts can also be identified. As shown in Figure 1, left lane, the tRNAVal (UAC) gene (wt) is expressed in vivo in yeast yielding primary transcripts which start at nucleotides -12 and -9. These primary transcripts are processed to tRNAs with mature 5' ends. Similarly, a variant of the tRNAval(UAC) gene, NF:8B, is expressed and processed in vivo in yeast, although with reduced efficiency (Figure 1, central lane). This drop in efficiency results from binding of a nuclear DNA binding protein which specifically recognizes the BamHI linker inserted at position -3 within the 5' flanking region of the tRNAval(UAC) region (Marschalek and
Dingermann, 1988). A remarkably different expression behaviour shows another tRNAval(UAC) variant gene, AN:8B. Although transcribed efficiently in vivo in yeast, primary transcripts accumulate and are not processed to mature size tRNAs (Figure 1, right lane). Plasmid AN:8B was fortuitously obtained during the cloning procedure of NF:8B and differs from NF:8B by a deletion of nucleotides -11, -3 and -2 in the wild-type 5' flanking region. Neither NF:8B nor AN:8B contains any alterations within the mature tRNA coding region or within the 3' flanking region with respect to the wild-type gene. The tRNAV8I(UAC) from D.discoideum contains an unusually labile amino acid acceptor stem It has been demonstrated that a highly ordered structure of primary transcripts and not any consensus sequence is responsible for recognition of tRNA processing enzymes (Garber and Altman, 1979; Nishikura et al., 1982, Baldi et al., 1983; Mattoccia et al., 1983; Traboni et al., 1984; Castano et al., 1985). The tRNAval(UAC) gene from D.discoideum codes for transcripts with a particularly unstable amino acid acceptor stem (see Figure 2a - c). In
addition to a G2 -U71 base pair there is also a G5 -G68 mismatch within this amino acid acceptor stem. Nevertheless, primary transcripts of the primary wild-type gene are processed correctly suggesting that the G5 -G68 mismatch plus the G2 -U71 base pair do not interfere with recognition requirements of processing enzymes. The processing deficiency of primary transcripts derived from AN:8B, however, points to the possibility that flanking regions might
1254
_ !Z
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_ "I
cI.D)' A r-'(S 1)P
I
(liI1il tl)
(I i II u
oI():
it
Alffi'mu 0 ;.
= tR\I V''k~
11
i;
t l
.D
4
40 ..
L
Fig. 1. Primer extension analysis of tRNAs synthesized in vivo in yeast from a D.discoideum tRNAval(UAC) gene (wt) and from derivatives NF:8B and AN:8B. Bulk tRNA was isolated from yeast strains which were transformed with plasmids carrying the indicated tRNA genes. A 32P-end-labelled oligonucleotide recognizing exclusively products of the tRNAVa (UAC) gene and of its derivatives was hybridized with 0.25 A260 units of the respective bulk tRNAs. cDNAs were synthesized and size-fractionated on sequencing gels. An extended primer fragment of 40 nucleotides corresponds to mature tRNA (indicated by the small box). Larger cDNAs were synthesized on primary transcripts while fragments
