Cell, Vol. 17, 399-406.
June
1979,
Copyright
0 1979 by MIT
In Vitro Transcription and Processing tRNA Gene Containing an Intervening
Richard C. Ogden, Jacques S. Beckman,* John Abelson and Hyen S. Kang Department of Chemistry University of California at San Diego La Jolla, California 92093 Dieter Soil and Otto Schmidt Yale University Box 1937, Yale Station New Haven, Connecticut 06520
Summary A gene for Saccharomyces cerevisiae tRNATW has been sequenced which contains an intervening sequence of 34 bp (H. S. Kang and J. Abelson, unpub iished results). The mutant yeast strain ts-136 accumulates a precursor to tRNATW which contains mature ends and is coiinear with the tRNATW gene. A nuclear extract from Xenopus oocytes is capable of supporting transcription of the tRNATW gene contained on piasmid pBR313. The products are precursor tRNAs which contain the intervening RNA sequence.The Xenopus extract accurately splices the precursor transcript to mature-sized tRNATW. introduction The detailed study of eucaryotic tRNA genes and of the synthesis and maturation of their products has just begun. individual eucaryotic tRNA genes have been isolated, identified and, in a few cases, sequenced (Goodman, Olson and Hail, 1977; Kressmann et al., 1976; Schmidt et al., 1976; Vaienzuela et al., 1976). Some of these tRNA genes were shown to be noncolinear with their products; they contained intervening nucieotide sequences which are removed at the RNA level by novel enzymes of RNA metabolism (Knapp et al., 1976; O’Farreii et al., 1978). Detailed studies of the complex enzymatic mechanism of eucaryotic tRNA biosynthesis necessitate the availability of the initial transcription products of tRNA genes; these RNAs would serve as substrates for the many enzymes involved in tRNA maturation. Since tRNA biosynthesis in lower eucaryotes proceeds generally very quickly, the level of tRNA precursors in normal cells is very low. A mutant strain of yeast OS-1 36) with altered RNA metabolism has been found to accumulate tRNA precursors (Hopper, Banks and Evangelidis, 1978). An examination of these precursors showed them to be a small class only; five of them were identified as pre-tRNATY’, pre-tRNAPh”, pre-tRNA%! , pre-tRNATrP and pre-tRNA!$“. They ail contain intervening sequences. Two of these RNAs, pre-tRNATY’ l Present address: Agricultural Research Organization, The Volcani Center, Institute of Field and Garden Crops, P.O.B. 6. Bet Dagan. Israel.
of a Yeast Sequence
and pre-tRNAPhe, were extensively characterized. The existence of intervening sequences for these genes had been predicted by DNA sequencing (Goodman et al., 1977; Vaienzueia et al., 1978). Knapp et al. (1978) and O’Farreii et al. (1978) demonstrated that these intervening sequences were transcribed in vivo. These precursors allowed the identification and characterization of a splicing enzyme which removes these additional nucleotides from within the sequence of the mature tRNA. We have earlier shown that a nuclear extract for Xenopus oocytes is capable of excellent transcription of Drosophila and Schizosaccharomyces pombe tRNA genes in vitro into tRNA precursors and tRNA species (Schmidt et al., 1978). Ultimately such a system should allow the characterization of transcription initiation and termination sites of RNA poiymerase iii and provide the desired tRNA precursors for the studies of tRNA maturation. in the present study, we tested this heteroiogous system for the transcription of an S. cerevisiae tRNATrp gene which contains a 34 nucieotide intervening sequence (H. S. Kang and J. Abelson, unpublished results). A comparison of the in vitro formed RNA products with the in vivo precursor from strain ts-136 showed that the Xenopus germinal vesicle (GV) extract accurately transcribes the yeast tRNATrP gene. The intervening sequences were found in the RNA precursors. Moreover, processing occurred in the heteroiogous system which resulted in accurate removal of the intervening sequences. Results Characterization of a tRNATW Precursor from in Vivo Labeled Yeast it has previously been reported that the yeast mutant ts-136 accumulates a precursor to tRNATrp at the nonpermissive temperature and that this precursor can be processed to 4S-sized material upon incubation with a crude extract from yeast (Knapp et al., 1978). The processing reaction involves the removal of about 35 nucieotides as judged by gel eiectrophoretie mobiiiity. The uniformly labeled precursor tRNA was purified by hybridization to piasmid DNA containing the tRNATP gene and analyzed by fingerprinting. As in the case of pre-tRNAPhe and pre-tRNATy’ (Knapp et al., 1978), the RNAase Tl and pancreatic RNAase fingerprints established the presence of mature 5’ and 3’ ends, but showed several additional oiigonucieotides which could not be assigned to the mature tRNA sequence (Keith et al., 1971). Further analysis of these oiigonucieotides allowed some sequence eiucidation and suggested that the additional RNA was inserted in the mature sequence adjacent to the 3’ end of the anticodon. The autoradiogram of the fingerprint from the complete digestion of pre-tRNATrP
Cell 400
with Tl RNAase and subsequent analysis of oligonucleotides are shown in Figure 1 and Table 1, respectively. Oligonucleotides corresponding to possible sequences beyond the termini are not present. Oligonucleotides Tl 1 and T19 span the junctions between the intervening sequence and the mature tRNA. Oligonucleotides T12, T14 and T16 are unique to the intervening sequence. Minor base analysis of total pre-tRNATrP and oligonucleotides produced by complete digestion with RNAase Tl or pancreatic RNAase demonstrated that although some modifications were present throughout the molecule in almost molar yield (for example, \k, D, T, m’G), others were totally absent (Gm. Cm) or present in low yield (m’G, m’ A). A qualitative summary of the minor base analysis is shown in Table 2. The DNA sequence of a tRNATrp gene from yeast confirmed the presence of an intervening sequence of 34 bases adjacent to the anticodon and indicated the primary structure of the tRNATW precursor (H. S. Kang and J. Abelson, unpublished results).
Transcription of the Yeast tRNATm Gene in Xenopus Nuclear Extracts A recombinant plasmid containing a single yeast tRNATrp gene has been previously isolated (Beckmann et al., 1977) and used to determine the sequence of the tRNATrp gene (H. S. Kang and J. Abelson, unpublished results). This plasmid DNA was transcribed in
an extract from Xenopus germinal vesicles (Birkenmeier, Brown and Jordan, 1978; Schmidt et al., 1978) and the RNA products were separated by polyacrylamide gel electrophoresis. Three major products, designated RNA-l, RNA-2 and RNA-3, were observed which migrate in the 4-5s region (Figure 2, lane 1). Transcription experiments with varying concentrations of a-amanitin (Schmidt et al, 1978) demonstrated that RNA polymerase Ill is responsible for the transcription of these RNA species. The stable RNA transcripts formed in the GV system Table
1. RNAasa
Tl Oligonucleotides
from in Vivo Pre-tRNAT’P
Yields (Molar) spot Number
Experimental
Tl
7.30
Theoretical
Identity’
June
1979,
Copyright
0 1979 by MIT
In Vitro Transcription and Processing tRNA Gene Containing an Intervening
Richard C. Ogden, Jacques S. Beckman,* John Abelson and Hyen S. Kang Department of Chemistry University of California at San Diego La Jolla, California 92093 Dieter Soil and Otto Schmidt Yale University Box 1937, Yale Station New Haven, Connecticut 06520
Summary A gene for Saccharomyces cerevisiae tRNATW has been sequenced which contains an intervening sequence of 34 bp (H. S. Kang and J. Abelson, unpub iished results). The mutant yeast strain ts-136 accumulates a precursor to tRNATW which contains mature ends and is coiinear with the tRNATW gene. A nuclear extract from Xenopus oocytes is capable of supporting transcription of the tRNATW gene contained on piasmid pBR313. The products are precursor tRNAs which contain the intervening RNA sequence.The Xenopus extract accurately splices the precursor transcript to mature-sized tRNATW. introduction The detailed study of eucaryotic tRNA genes and of the synthesis and maturation of their products has just begun. individual eucaryotic tRNA genes have been isolated, identified and, in a few cases, sequenced (Goodman, Olson and Hail, 1977; Kressmann et al., 1976; Schmidt et al., 1976; Vaienzuela et al., 1976). Some of these tRNA genes were shown to be noncolinear with their products; they contained intervening nucieotide sequences which are removed at the RNA level by novel enzymes of RNA metabolism (Knapp et al., 1976; O’Farreii et al., 1978). Detailed studies of the complex enzymatic mechanism of eucaryotic tRNA biosynthesis necessitate the availability of the initial transcription products of tRNA genes; these RNAs would serve as substrates for the many enzymes involved in tRNA maturation. Since tRNA biosynthesis in lower eucaryotes proceeds generally very quickly, the level of tRNA precursors in normal cells is very low. A mutant strain of yeast OS-1 36) with altered RNA metabolism has been found to accumulate tRNA precursors (Hopper, Banks and Evangelidis, 1978). An examination of these precursors showed them to be a small class only; five of them were identified as pre-tRNATY’, pre-tRNAPh”, pre-tRNA%! , pre-tRNATrP and pre-tRNA!$“. They ail contain intervening sequences. Two of these RNAs, pre-tRNATY’ l Present address: Agricultural Research Organization, The Volcani Center, Institute of Field and Garden Crops, P.O.B. 6. Bet Dagan. Israel.
of a Yeast Sequence
and pre-tRNAPhe, were extensively characterized. The existence of intervening sequences for these genes had been predicted by DNA sequencing (Goodman et al., 1977; Vaienzueia et al., 1978). Knapp et al. (1978) and O’Farreii et al. (1978) demonstrated that these intervening sequences were transcribed in vivo. These precursors allowed the identification and characterization of a splicing enzyme which removes these additional nucleotides from within the sequence of the mature tRNA. We have earlier shown that a nuclear extract for Xenopus oocytes is capable of excellent transcription of Drosophila and Schizosaccharomyces pombe tRNA genes in vitro into tRNA precursors and tRNA species (Schmidt et al., 1978). Ultimately such a system should allow the characterization of transcription initiation and termination sites of RNA poiymerase iii and provide the desired tRNA precursors for the studies of tRNA maturation. in the present study, we tested this heteroiogous system for the transcription of an S. cerevisiae tRNATrp gene which contains a 34 nucieotide intervening sequence (H. S. Kang and J. Abelson, unpublished results). A comparison of the in vitro formed RNA products with the in vivo precursor from strain ts-136 showed that the Xenopus germinal vesicle (GV) extract accurately transcribes the yeast tRNATrP gene. The intervening sequences were found in the RNA precursors. Moreover, processing occurred in the heteroiogous system which resulted in accurate removal of the intervening sequences. Results Characterization of a tRNATW Precursor from in Vivo Labeled Yeast it has previously been reported that the yeast mutant ts-136 accumulates a precursor to tRNATrp at the nonpermissive temperature and that this precursor can be processed to 4S-sized material upon incubation with a crude extract from yeast (Knapp et al., 1978). The processing reaction involves the removal of about 35 nucieotides as judged by gel eiectrophoretie mobiiiity. The uniformly labeled precursor tRNA was purified by hybridization to piasmid DNA containing the tRNATP gene and analyzed by fingerprinting. As in the case of pre-tRNAPhe and pre-tRNATy’ (Knapp et al., 1978), the RNAase Tl and pancreatic RNAase fingerprints established the presence of mature 5’ and 3’ ends, but showed several additional oiigonucieotides which could not be assigned to the mature tRNA sequence (Keith et al., 1971). Further analysis of these oiigonucieotides allowed some sequence eiucidation and suggested that the additional RNA was inserted in the mature sequence adjacent to the 3’ end of the anticodon. The autoradiogram of the fingerprint from the complete digestion of pre-tRNATrP
Cell 400
with Tl RNAase and subsequent analysis of oligonucleotides are shown in Figure 1 and Table 1, respectively. Oligonucleotides corresponding to possible sequences beyond the termini are not present. Oligonucleotides Tl 1 and T19 span the junctions between the intervening sequence and the mature tRNA. Oligonucleotides T12, T14 and T16 are unique to the intervening sequence. Minor base analysis of total pre-tRNATrP and oligonucleotides produced by complete digestion with RNAase Tl or pancreatic RNAase demonstrated that although some modifications were present throughout the molecule in almost molar yield (for example, \k, D, T, m’G), others were totally absent (Gm. Cm) or present in low yield (m’G, m’ A). A qualitative summary of the minor base analysis is shown in Table 2. The DNA sequence of a tRNATrp gene from yeast confirmed the presence of an intervening sequence of 34 bases adjacent to the anticodon and indicated the primary structure of the tRNATW precursor (H. S. Kang and J. Abelson, unpublished results).
Transcription of the Yeast tRNATm Gene in Xenopus Nuclear Extracts A recombinant plasmid containing a single yeast tRNATrp gene has been previously isolated (Beckmann et al., 1977) and used to determine the sequence of the tRNATrp gene (H. S. Kang and J. Abelson, unpublished results). This plasmid DNA was transcribed in
an extract from Xenopus germinal vesicles (Birkenmeier, Brown and Jordan, 1978; Schmidt et al., 1978) and the RNA products were separated by polyacrylamide gel electrophoresis. Three major products, designated RNA-l, RNA-2 and RNA-3, were observed which migrate in the 4-5s region (Figure 2, lane 1). Transcription experiments with varying concentrations of a-amanitin (Schmidt et al, 1978) demonstrated that RNA polymerase Ill is responsible for the transcription of these RNA species. The stable RNA transcripts formed in the GV system Table
1. RNAasa
Tl Oligonucleotides
from in Vivo Pre-tRNAT’P
Yields (Molar) spot Number
Experimental
Tl
7.30
Theoretical
Identity’
