Transcription of aDrosophilatRNA^Arggene in yeast extract: 5′-flanking sequence dependence for transcription in a heterologous system

Abstract

The Drosophila tRNA gene encoded on pArg is efficiently transcribed in extracts of Saccharomyces cerevisiae, but the efficiency is 5'-flanking sequence dependent: deletion to between positions -21 and -17 (relative to position +1 of the mature coding sequence) reduces transcription to a very low level. This demonstrates that requirement for wild-type 5'-flanking sequence exists in the case of a heterologous combination of a tRNA gene and transcription extract. Expression of pArg in vivo in S. cerevisiae is also dependent on the wild-type 5'-flanking sequence, but only with deletion to between -17 and -11 is the steady-state level of pArg transcripts reduced to near zero. The 5'-flanking sequence requirement in S. cerevisiae extract is similar to that found in Drosophila Kc cell extract. However, transcription kinetics distinguish S. cerevisiae extract from that of Drosophila Kc cells. tRNA genes added to S. cerevisiae extract exhibit a lag phase before initiation of active transcription, but this lag is much shorter and much less temperature dependent than is the lag phase in Drosophila Kc cell extract.

Expression of nuclear tRNATyr genes from Arabidopsis thaliana in HeLa cell and wheat germ extracts

“…Since a homologous in vitro transcription system of plant origin is not available for RNA polymerase III-transcribed genes, we have used a HeLa cell nuclear extract for studying the expression of the Arabidopsis tRNA xyr genes. HeLa cell extracts have proved to be an almost universal transcription system for tRNA genes from organisms as unrelated as yeast [22], Drosophila [19] and Xenopus [7,11 ]. We have shown that an intron-containing tRNA Ty~ gene (pNtY 1) isolated from tobacco (Nicotiana rustica) is also efficiently transcribed and accurately processed and spliced in HeLa extract [27 ].…”

An upstream signal is required for in vitro transcription ofNeurospora 5S RNA genes

“…1981Dingermann et al 1983;Galli et al 1981;Wilson et al 1985;Enver 1985;Segall et al 1980;Shastry et al 1982) and to the characterization of proteins that specifically bind to them (Enver 1985;Segall 1980;Shastry et al 1982). Although transcription of genes by pol III in some in vitro systems is solely dependent on internal promoter regions, evidence for both stimulation (Cooley et al 1984;Johnson and Raymond 1984;Larson et al 1983;Indik and Tartof 1982;Schaack et al, 1984;Schaack and $611 1985;Shaw and Olson 1984;Sprague etal. 1980) and inhibition (DeFranco et al 1980(DeFranco et al , 1981Hipskind and Clarkson 1983) of transcription by sequences immediately upstream of coding regions has been reported.…”

“…It will be interesting to learn whether these sequences share a function, such as in polymerase binding. It must be stressed, however, that many polymerase III templates lack the TATA box, and some of these require other flanking sequences for transcription (Cooley et al 1984;Schaack et al 1984;Schaack and $611 1985). Different requirements for upstream sequences may reflect different control mechanisms required for developmental or tissue-specific regulation.…”

Dimeric tRNA gene arrangement inSchizosaccharomyces pombeallows increased expression of the downstream gene

“…Thus, the occurrence of dimeric tRNA genes in a eukaryote involves the duplication of promoter sequences, and raises the questions of why the duplication of promoter regions is required and tolerated, and whether the promoter elements of both genes in the dimer are functional. Transcription of tRNA genes in S. cerevisiae has been shown to require an appropriate 5'-flanking sequence (14)(15)(16)(17). Further, the mechanism of eukaryotic tRNA gene transcription involves the stable binding © I RL Press Limited, Oxford, England.…”