Promoter of a eukaryotic tRNAPro gene is composed of tree noncontiguous regions.

Abstract

The 71-base-pair coding sequence ofthe tRNAP1O gene from Caenorhabditis elegans contains all of the information required for transcription and processing in the injected oocytes.Several subclones of the DNA coding for the tRNAP"' were constructed, carrying deletions or insertions, or both. Their transcriptional properties lead to the hypothesis that the tRNAPW gene promoter is composed of three discontinuous regions within the coding sequence.In previous studies we have characterized steps in the synthesis of mature tRNA molecules in eukaryotic cells by injecting cloned DNAs coding for tRNA into Xenopus laevis oocytes (1-4). These studies have shown that tRNA genes are transcribed into precursor molecules, which are converted into tRNAs through a series of nucleolytic cleavages and nucleotide modifications.In order to gain further insight into the process of tRNA biosynthesis and to determine how cells regulate the expression of these genes, it will be instructive to identify the transcriptional signals present on the DNA sequence and recognized by the RNA polymerase III. The work ofBrown and his colleagues (5, 6) has revealed that the relatively small 5S RNA gene contains a 30-base-pair region within the coding sequence that is essential for initiation of transcription by RNA polymerase III. Also for tRNA genes (7-10), it appears probable that their promoters are not located in the 5' flanking region, even though this region may play a regulatory role in some cases (9, 10). It is unclear, however, whether they have an internal control region analogous to that of the 5S RNA genes. To establish this, we constructed deletion and insertion mutants, altering sequences within the coding regions of the tRNAP1O gene of Caenorhabditis elegans. This allowed the identification ofthree separated regions within the coding sequence that are important for transcription of the tRNAPr' gene. MATERIALS AND METHODSChemicals and Enzymes. Isopropyl-D-thiogalactopyranoside and 5-bromo-4-chloro-3-indolyl-D-galactoside were purchased from Sigma, and recombinant DNA linkers were purchased from Collaborative Research (Waltham, MA). T4 DNA ligase, polynucleotide kinase, and endonucleases EcoRI and Hae III were gifts of V. Pirrotta. Endonucleases HindIII and Sin I and DNA polymerase large fragment Klenow enzyme were purchased from Boehringer. Endonucleases HinfI and BamHI and nucleases S1 and BAL 31 were purchased from Bethesda Research Laboratories. Exonuclease Xma was a gift of M. Zabeau. All radioactive compounds were purchased from Amersham Buchler, Braunschweig.Bacterial Strains, Plasmids, and Cloning Vehicles. Escherichia coli K-12 (strain 71-18) was used for transformation with phage M13 (strain mp2) replicative form I and all of its recombinant derivatives (4, 11, 12). E. coli K-12 (strain K514) was used for transformation with pBR322 plasmid and its recombinantderivatives Protocolsfortransformation and preparation of double-stranded DNA were as described (1, 4).Polyacrylamide Gel Electrophoresis. DNA restriction fragment...

Transcripts of mitochondrial tRNA genes inSaccharomyces cerevisiae

“…This model would imply that, at least for this group of genes, transcriptionwould not require internal promotors present in each tRNA gene as has been demonstrated for several eukaryotic tRNAs (24,25,26); the synthesis of this group of tRNAs would actually involve, as in mitochondria from HeLa cells (27), the processing of longer transcripts.…”

Relationship between the two components of the split promoter of eukaryotic tRNA genes.

“…In this figure we also plotted the extent of transcription obtained with two deletion mutants that actually shorten the distance between the essential regions and that have been characterized elsewhere (6) (19). Mcetl and pBcetl91 have been described (6,12). The other represented plasmids were constructed in the following manner: pBcet74: Mcet7 DNA was digested with restriction endonuclease Hha I followed by S1 nuclease, to generate blunt ends.…”

“…We fractionated these by 10% gel electrophoresis and purified the EcoRI/EcoRI fragment of 320 bp, corresponding to the hybrid gene. This was then inserted into pACYC184 plasmid (20) The starting plasmids were: Mcet7, carrying a 250-bp nematode insert into the BamHI site ofphage mpLL2 (19) and coding for a tRNALU (unpublished data), and Mcetl, coding for a tRNAP', which has been described (6,12). The cloning strategy is represented diagrammatically in Fig.…”

“…2), in which the nematode tRNA sequences are cloned in pBR322 between the EcoRI and the BamHI sites, with a HindIII linker separating the coding region from the 3' flanking sequence. The tRNAPrO gene inserted in this clone has a deletion of the 5' flanking sequence; however, this deletion does not affect the transcriptional activity of the gene (6). The construction of insertion mutants (illustrated in Fig.…”

“…In the VAI RNA gene of adenovirus an internal control region, approximately 60 bp long, could be identified (3,4). In the case oftRNA genes a more precise analysis leads to the identification of two sequences of about 10 nucleotides each, located within the coding region, whose presence is essential for transcription (5,6). This overlap of coding sequences and transcriptional signals is characteristic of eukaryotic organisms: the corresponding genes (5S RNA, tRNA) in prokaryotes have a classical promoter clearly separated from the coding region and located in the 5' flanking region (7).…”