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Eukaryotic Trna Genes (Hybrid Gene/Insertion Mutants/Nuclear Microinjection/RNA Polymerase HI Transcription) G

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Eukaryotic Trna Genes (Hybrid Gene/Insertion Mutants/Nuclear Microinjection/RNA Polymerase HI Transcription) G Proc. NatL Acad. Sci. USA Vol. 79, pp. 1921-1925, March 1982 Genetics Relationship between the two components of the split promoter of eukaryotic tRNA genes (hybrid gene/insertion mutants/nuclear microinjection/RNA polymerase HI transcription) G. CILIBERTO*, C. TRABONI*, AND R. CORTESE European Molecular Biology Laboratory, Postfach 102209, 6900 Heidelberg, Federal Republic of Germany Communicated by Sydney Brenner, October 21, 1981 ABSTRACT Plasmids containing eukaryotic tRNA genes are stitute the split promoter of a tDNAPro from Caenorhabditis faithfully transcribed in the nucleus of Xenopus loevis oocytes elegans (6) and of a tDNAMet from Xenopus laevis (5). [Cortese, R., Melton, D. A., Tranquilla, T. & Smith, J. D. (1978) In the light of this idea one can recognize at least two other Nucleic Acids Res. 5, 4593-4611]. It has been established that two structural features that must be common to all tRNA genes. One separated regions within the coding sequence of a tRNA gene are is the physical distance between the two invariant regions: this essential and sufficient for promotion oftranscription [Hofstetter, is about 40 nucleotides in almost all tRNAs, with the exception H., Kressmann, A. & Birnstiel, M. L. (1981) Cell 24, 573-585; of those tRNA genes that contain introns. The other common Ciliberto, G., Castagnoli, L., Melton, D. A. & Cortese, R. (1982) feature is the Proc. Natl. Acad. Sci. USA 79, 1195-1199]. We have constructed overall partial self-complementarity ofthe 5' and a hybrid tRNA gene containing one essential region from tDNAIeU 3' halves ofa tRNA molecule, which confers on the correspond- and the other from tDNAPrO, both from Caenorhabditis elegans. ing DNA sequence the potential for folding into a cloverleaf-like This hybrid gene is efficiently transcribed, thus showing that the secondary structure. We asked ourselves ifany ofthese features essential regions are independent transcriptional signals regard- play a role in transcription. less of the overall regularities of the structure oftRNA genes. We In order to provide an answer to these questions we have have also constructed mutants of the tRNA"' gene in which the constructed two different sets of mutant tRNA genes. In one distance between the two essential regions is changed; optimal set we varied the distance between the two essential regions; transcription occurs when this distance is about40-50 nucleotides. in the other we recombined "essential regions" so as to obtain a new "gene" whose promoter is composed ofa 5' halfderiving It has been firmly established that the genes transcribed by from tDNALu and the 3' half from tDNAPr, from C. elegans RNA polymerase III contain their transcriptional signals (pro- (tDNA is the DNA coding for tRNA). moters) within the coding sequence. In 5S RNA genes a 30-base pair (bp) region seems to contain all the information necessary EXPERIMENTAL PROCEDURES for correct initiation oftranscription (1, 2). In the VAI RNA gene Recombinant DNA work was performed according to the rec- of adenovirus an internal control region, approximately 60 bp ommendations outlined in the National Institutes of Health long, could be identified (3, 4). In the case oftRNA genes a more guidelines ofJune 23, 1976. precise analysis leads to the identification of two sequences of Purification of Flush-End DNA Fragments from Plasmid about 10 nucleotides each, located within the coding region, pBR322. Double-stranded pBR322 DNA was cut either with whose presence is essential for transcription (5, 6). This overlap restriction endonuclease Alu I (Boehringer) or with restriction ofcoding sequences and transcriptional signals is characteristic endonuclease Hae III (gift ofV. Pirrotta). DNA restriction frag- of eukaryotic organisms: the corresponding genes (5S RNA, ments were separated electrophoretically on Tris glycine gels tRNA) in prokaryotes have aclassical promoter clearly separated (11). The section of the gel containing fragments 19 to 201 bp from the coding region and located in the 5' flanking region long was cut out and the fragments were eluted as described (7). Because the structure oftRNA is very similar in eukaryotes elsewhere (12). and prokaryotes (8), the obvious conclusion is that the DNA Construction of Insertion Mutants. Plasmid pBcetl3 was coding sequences have not been modified to be adapted to the first cut with the restriction endonuclease Sma I (purchased new function of transcriptional signals, but rather the opposite from Boehringer) and then ligated in the presence ofeach ofthe must have occurred, namely that the transcriptional machinery previously purified flush-end DNA fragments from pBR322. Phage T4 DNA ligase was a gift of V. Pirrotta. has evolved the capacity to recognize parts of the coding se- Escherichia coli K-12, strain Hb 101, was used for transfor- quence as transcriptional signals. Because all tRNA genes are mation. Protocols for transformation and preparation ofdouble- transcribed by RNA polymerase III (9) it is reasonable to expect stranded DNA were as described (11). Transformants grown on that the transcriptional signals must correspond to features com- LB plates supplemented with ampicillin at 100 Ag/ml were mon to all tRNA genes. One of these features is the so-called screened for the presence of the inserted fragment. This was invariant nucleotides. On the basis of the analysis of several done by rapidly preparing plasmids on a microscale (13) and mutants of a tRNATYr gene of yeast Koski et al. (10) have hy- then lookingfor the loss ofthe Sma I recognition site. The length pothesized that the internal promoter of a tRNA gene is essen- of the insert was established by comparing the total length of tially constituted by these invariant nucleotides. It is interesting the coding region in the wild-type gene (plasmid pBcetl3) and that these invariant nucleotides are particularly, although not in the insertion mutants, after digestion with restriction en- exclusively, concentrated in the two essential regions that con- donucleases HindIII and BamHI (purchased from Bethesda The publication costs ofthis article were defrayed in part by page charge Abbreviations: bp, base pair(s); tDNA, DNA coding for tRNA. payment. This article must therefore be hereby marked "advertise- * On leave of absence from Istituto di Chimica Biologica, II Facolta di ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. Medicina, University of Naples, Naples, Italy. 1921 Downloaded by guest on September 28, 2021 1922 Genetics: Ciliberto et aL Proc. Natl. Acad. Sci. USA 79 (1982) Research Laboratories). DNA polymerase large fragment (Kle- now enzyme) used to end-label DNA fragments was purchased from Boehringer. Construction of a tDNAI"U-1)NAP' Recombinant Gene. p~~cetl3~~PBR322 The parental plasmids were Mcet7, containing a 250-bp nem- p~~cetl3~~cut withAlu "I atode DNA insert with a single copy of a tDNAJeU from C. \ / \ \ ~~~~~~~orHae HI / elegans and Mceti, containing a 263-bp nematode DNA insert /EcoRI with a single copy ofa tDNAP"". The vectorwas phage mp2 (14). tDNA\~\ tDNAF'ror~ _o All the steps needed for the construction ofthe hybrid gene are BamHI _ HindJII represented in Fig. 5. Recombinant DNA linkers were pur- Sma I chased from Collaborative Research, Waltham, MA. Endonu- Cut with Sma I Blunt-end ligation of pBcetl3 clease Hha I and S1 nuclease were purchased from Bethesda with purified fragments of Research Laboratories. pBR322. Transformation, Microinjection and Quantitation Analysis. Microinjections purification of cloned DNA, into the nuclei ofXenopus oocytes were performed as described digestion with BamHI and (11). All plasmid DNAs were injected at the concentration of Hindll terminal labeling. 250 ,tg/ml. RNA transcripts were separated electrophoretically with 32 on 5% or 8% polyacrylamide gels run in TBE buffer (11). Quan- titative analysis was performed as follows: Bands were cut and 1 2 3 4 5 G T 8 9 10 ii _ . __ their radioactivities were measured as Cerenkov counts. A nor- ON. - __d - malization was then done, using as internal standard the radio- 4 activity incorporated-in the endogenous 18- and 28S rRNA spe- cies. This was normally done by a previous fractionation of a separate aliquot on a 2% agarose gel followed by measurement 4 of the radioactivity contained in the gel slices. In practice we 4 found that identical values were obtained also when, rather than 4i4 fractionating the rRNA on a separate gel, we normalized to the b radioactivity present at the top ofthe polyacrylamide gels. We I chose 18- and 28S rRNA as internal standard rather than 5S RNA because in this smaller molecule there was little incorporation 4 I ofradioactive precursors. We discarded the alternative ofcoin- jecting a purified 5S RNA gene, as was done by Hofstetter et al. (5), because we found that there is a considerable degree of -45>5iInr s~ competition with tRNA genes (15). FIG. 2. (Upper) Construction of insertion mutants of tDNA~'. Radioactive Compounds and Autoradiography. All radio- (Lower) Ten percent polyacrylamide gel electrophoresis of the end-la- active compounds were purchased from Amersham Buchler, beledHindl/BamHI DNA segments from the wild-type gene and the Braunschweig. Fuji films were used, occasionally with preflash- various insertion mutants: lanes 1 and 11, end-labeled Hinfl digest of ing according to Laskey and Mills (16). pBR322 as size marker; lane 2, pBcetl3 (wild-type gene); lane 3, pBcetl3/1; lane 4, pBcetl3/2; lane 5, pBcetl3/3; lane 6, pBcetl3/4; lane 7, pBcetl3/5; lane 8, pBcetl3/6; lane 9, pBcetl3/7; lane 10, RESULTS pBcetl3/8. Mutants carry the following segments from pBR322 (17): pBcetl3/1, 2117-2135; pBcetl3/2, 2068-2116; pBcetl3/3, 3656-3718; Construction of Insertion Mutants of a tRNAPrO Gene. In pBcetl3/4, aduplication offiagment 2068-2116, pBcetl3/5, 3556-3655; a previous study of the structure of the promoter of a tRNAPrO pBcetl3/6, 2643-2778; pBcetl3/7, 1262-1445; pBcetl3/8, 3556-3756. gene from C.
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