Two control regions for eukaryotic tRNA gene transcription.

Abstract

Two Drosophila tRNALYS genes with identical coding sequences were shown to transcribe with very different efficiencies in nuclear extracts from Xenopus oocytes. The use of recombinant plasmids in which the 5'-flanking sequences of these genes were either "switched" or replaced by defined pBR322 sequences revealed two control regions for tRNA gene transcription. An internal control region comprising the mature tRNA coding sequence (and possibly its 3'-flanking sequences) is sufficient for transcription initiation, and an external control region comprising the 5'-flanking sequences represses this transcription. All transcripts have short leader sequences. Altered precursor tRNAs transcribed from truncated tRNALYS genes (missing a single base pair in the acceptor stem) are not processed well in vitro.Attempts to elucidate the signals controlling eukaryotic gene transcription by RNA polymerase III (1) have been greatly facilitated by the availability of genes of known DNA sequence that can be transcribed faithfully. Injection of cloned genes for Xenopus 5S RNA (2), Xenopus initiator tRNA (3), and yeast tRNA (4) into the Xenopus oocyte nucleus results in the correct transcription of these genes into the mature RNA. The use of nuclear extracts from Xenopus oocytes has allowed in vitro transcription of Xenopus 5S RNA (5) or Drosophila, yeast, and Bombyx tRNA genes (6-9). Such in vitro experiments have defined initiation and termination sites for RNA transcription in Xenopus 5S RNA (10), Drosophila tRNAArg (11), and Bombyx tRNAAla (8,9).Comparison of the DNA sequences of several Xenopus 5S RNA genes revealed a conserved nucleotide sequence in the 5'-flanking region of the mature 5S RNA coding sequence (12). However, the results of RNA transcription experiments using 5S genes with shortened 5'-leader regions suggested that these conserved sequences are not important for initiation of 5S gene transcription (13). Experiments with enzymatically truncated genes had an unexpected result: the sole requirement for proper transcription of 5S or 5S-size RNA is the maintenance of the gene segment between nucleotides +50 and +80 of the mature 5S RNA coding sequence (14,15).Sequence analysis of various eukaryotic tRNA genes (7, 16-19) revealed no obvious common "promoter-like" sequence in the 5'-flanking regions. Moreover, a tRNA gene that has only 10 nucleotides upstream of the RNA initiation site transcribes well (8, 9). However, because the protein initiation factors required for 5S DNA transcription are different from those for tRNA genes (20), the DNA regions controlling transcription may not be the same for 5S and tRNA genes. This is also suggested by recent experiments showing that both the anterior and posterior portions of a Xenopus tRNAMet gene unit are required for its proper transcription (21).Analysis of Drosophila tRNA genes gave the nucleotide sequences of four genes coding for tRNA2LYs (ref 19; this report).A sequence comparison (22) of the 5'-flanking regions of these genes revealed a highly conserved undec...

Transcription of a silkworm tRNAcAlagene is directed by two AT-rich upstream sequence elements

“…Upstream sequences that act negatively have also been identified. (11,13,12,14,10). Thus, the functional significance of upstream sequences is not in doubt.…”

“…Moreover, flanking sequences must be responsible for the fact that members of tRNA gene families that contain identical coding sequences frequently exhibit different transcription efficiencies. In several such cases, the dependence of transcription efficiency on upstream sequences has been demonstrated directly (11,12). Upstream sequences that act negatively have also been identified.…”

The expression of prokaryotic tRNA genes in frog oocytes

“…A unique feature of the mechanism of Pol III transcription is that the signals on the DNA required for transcription initiation are located within the coding portion of the gene (2)(3)(4)(5)(6)(7)(8). This is different from the transcription mechanism of prokaryotic genes where the promoter sequence lies at the 5' side of the coding sequence and is never transcribed into RNA (9).…”

“…A third tRNA gene (tRNA4eu) is transcribed at a considerably reduced rate. Our experiments didn't allow us to determine whether the reduced transcription is caused by weakness of the intragenic promoter or by a repressing effect of the 5'-flanking sequence (4). The behavior of the fourth tRNA gene (tRNAHis) is particularly perplexing.…”

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

“…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)(8)(9)(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.…”