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

Abstract

Three Schizosaccharomyces pombe dimeric tRNA genes, consisting of a tRNASer gene encoding a minor species with an intervening sequence followed by a tRNAMeti gene, have been described [Mao et al. (1980) Cell 21, 509-516; Hottinger et al. (1982) Mol. Gen. Genet. 188, 219-224; Willis et al. (1984) EMBO J. 3, 1573-1580]. We have examined the reason for the dimeric structure by comparing the transcriptional efficiencies and competitive abilities of the genes subcloned from the dimeric arrangement. Both of the subcloned genes are active in vivo in Saccharomyces cerevisiae, but only the tRNASer gene is efficiently transcribed in vitro. The tRNASer gene competes efficiently for transcription factors, while the tRNAMeti gene does so only weakly. Thus, it appears that the dimeric arrangement is required to support expression of the tRNAMeti gene. S. pombe genes encoding major species of tRNASer are transcribed considerably less efficiently than are the minor genes from the dimers, so coupling of the tRNAMeti gene to the minor species genes should lead to efficient production of tRNAMeti.

Local features determine Ty3 targeting frequency at RNA polymerase III transcription start sites

“…These tDNAs-tD(GUC)B, tD(GUC)D, tD(GUC)J2, and tD(GUC)J3-occur immediately downstream from tR(UCU) tDNAs-tR(UCU)B, tR(UCU)D, tR(UCU)J1, and tR(UCU)J2, respectively. Previous in vivo and in vitro evidence suggests that these tDNAs are expressed as dimeric transcripts (Kjellin-Straby et al 1984;Engelke et al 1985;Hottinger-Werlen et al 1985). In each case, the upstream tR(UCU) gene showed a far greater number of insertions than the downstream tD(GUC) gene, and insertions at the tD(GUC) gene could not be specifically assigned.…”

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

“…The transcription, of eukaryotic tRNA genes is directed by two internal control regions (1)(2)(3)(4)(5). Transcriptional efficiency in vitro can be modulated by 5'-flanking sequence elements (6)(7)(8)(9)(10). Extracts of Bombyx mori (11,12), Drosophila Kc (2,10,13), and Saccharomyces cerevisiae (14)(15)(16) cells have been shown to have a much greater positive requirement for 5'-flanking sequence than have extracts of Xenopus laevis germinal vesicles (1,3,11) or HeLa cells (2,10,13).…”

“…Transcriptional efficiency in vitro can be modulated by 5'-flanking sequence elements (6)(7)(8)(9)(10). Extracts of Bombyx mori (11,12), Drosophila Kc (2,10,13), and Saccharomyces cerevisiae (14)(15)(16) cells have been shown to have a much greater positive requirement for 5'-flanking sequence than have extracts of Xenopus laevis germinal vesicles (1,3,11) or HeLa cells (2,10,13). This led to the suggestions that a positive requirement for of a heterologous system, that of a Drosophila tRNAArg gene in S. cerevisiae extract, yields dependence on the wild-type 5'-flanking sequence, and that the extent of the dependence is in good agreement with that found using S. cerevisiae tRNA genes.…”

A mutation in the second largest subunit of TFIIIC increases a rate-limiting step in transcription by RNA polymerase III.

“…(19). TFIIIC activity was monitored by a mobility shift assay using a 32P-labeled 375-bp HindIII fragment containing the sup3-eST gene (16). Binding reactions in 20 ,ul contained 10 mM HEPES-KOH (pH 7.9), 10 mM MgCl2, 0.1 mM EDTA, 10% glycerol, 100 mM NaCl, 1 mM dithiothreitol 1 ,g of poly(dI-dC) poly(dIdC) (Pharmacia), the DNA probe (2,000 to 7,000 cpm/ng), and TFIIIC fractions and were incubated for 5 min at 25°C before electrophoresis (27).…”