RNA polymerase III catalysed transcription can be regulated in Saccharomyces cerevisiae by the bacterial tetracycline repressor-operator system.

Dingermann, Theo; Frank-Stoll, U; Werner, Herbert; Wißmann, Andreas; Hillen, Wolfgang; Jacquet, Michel; Marschalek, Rolf

doi:10.1002/j.1460-2075.1992.tb05193.x

Cited by 64 publications

(35 citation statements)

References 57 publications

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“…Therefore, we used a ''foreign'' tRNA. The D. discoideum intronless gene encodes a UAG nonsense suppressor tRNA Glu (tRNA Glu-D ) that is transcribed and processed into a tRNA that participates in translation in S. cerevisiae (21). We expressed the tRNA Glu-Dencoding gene from a centromere-containing plasmid, pRS416tRNA Glu-D , that is maintained in approximately one copy per nucleus (22) to assure a low rate of exchange of the DNA encoding tRNA Glu-D between heterokaryon nuclei.…”

Section: Resultsmentioning

confidence: 99%

Retrograde movement of tRNAs from the cytoplasm to the nucleus in Saccharomyces cerevisiae

Shaheen

Hopper

2005

Proc. Natl. Acad. Sci. U.S.A.

158

275

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In eukaryotes, tRNAs transcribed in the nucleus function in cytoplasmic protein synthesis. The Ran-GTP-binding exportin, Los1p͞ Xpo-t, and additional pathway(s) mediate tRNA transport to the cytoplasm. Although tRNA movement was thought to be unidirectional, recent reports that yeast precursor tRNA splicing occurs in the cytoplasm, whereas fully spliced tRNAs can reside in the nucleus, require that either the precursor tRNA splicing machinery or mature tRNAs move from the cytoplasm to the nucleus. Our data argue against the first possibility and strongly support the second. Combining heterokaryon analysis with fluorescence in situ hybridization, we show that a foreign tRNA encoded by one nucleus can move from the cytoplasm to a second nucleus that does not encode the tRNA. We also discovered nuclear accumulation of endogenous cytoplasmic tRNAs in haploid yeast cells in response to nutritional deprivation. Nuclear accumulation of cytoplasmic tRNA requires Ran and the Mtr10͞Kap111 member of the importin-␤ family. Retrograde tRNA nuclear import may provide a novel mechanism to regulate gene expression in eukaryotes.amino acid deprivation ͉ heterokaryon ͉ Mtr10 ͉ tRNA nuclear import ͉ yeast E ukaryotic precursor tRNAs (pretRNAs) contain 5Ј and 3Ј extra sequences and, for Ϸ25% of Saccharomyces cerevisiae (yeast) tRNA families, introns. To be fully functional, pretRNAs are subject to several maturation steps, including removal of the extra sequences, numerous nucleoside modifications, and addition of the 3Ј terminal CCA nucleotides (1). In vertebrates, nearly all tRNA posttranscriptional processing occurs in the nucleus before export of tRNAs to the cytoplasm (1-3). In contrast, several lines of evidence show that in yeast, pretRNA intron removal occurs in the cytoplasm, not the nucleoplasm. First, the subunits of the heterotetrameric tRNA-specific splicing endonuclease, Sen15, Sen2, Sen34, and Sen54, colocalize with mitochondria (4, 5). Tethering of a noncatalytic subunit, Sen54, to mitochondria does not inhibit pretRNA splicing, whereas mutations prohibiting its mitochondrial association cause accumulation of unspliced pretRNAs (4). Moreover, cells with conditional mutations in a gene encoding a catalytic subunit, Sen2, accumulate unspliced pretRNAs in the cytoplasm (4). A previous report employing immunofluorescence and immune electron microscopy concluded that a large fraction of tRNA ligase, catalyzing second step of pretRNA splicing, resides in the nucleus (6); however, more recent reports show that there is a functional pool of tRNA ligase activity in the cytoplasm (7) and that carboxyl GFP-tagged ligase is cytosolic (5). Finally, a carboxyl GFP-tagged version of the enzyme catalyzing the third step of pretRNA splicing, removal of the residual 2Ј phosphate (8), is distributed throughout the cytoplasm (5).Splicing of pretRNAs in the cytoplasm provides an explanation for the pretRNA splicing defects observed when there are mutations in the tRNA nuclear export machinery, including the tRNA exportin, Los1p (9-11), ...

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Section: Resultsmentioning

confidence: 99%

Retrograde movement of tRNAs from the cytoplasm to the nucleus in Saccharomyces cerevisiae

Shaheen

Hopper

2005

Proc. Natl. Acad. Sci. U.S.A.

158

275

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“…It should be mentioned that the tetracycline-inducible system has been used in several organisms, including Gram-positive bacteria, yeast, and fungi. [15][16][17][18] All these works focused their attention on pathogenic strains against which new antimicrobial agents should be developed.…”

Section: Discussionmentioning

confidence: 99%

Amplified Detection of Transcriptional and Translational Inhibitors in Bioluminescent Escherichia coli K-12

Galluzzi

Karp

2003

SLAS Discovery

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The excessive prescription of antimicrobial agents and their use as animal growth promoters lead to the spread of resistance among pathogenic bacteria. Consequently, unnecessary use should be minimized, and new chemicals with novel mechanisms of action are needed. The authors have developed a fast method to measure the activity of antibiotics by means of a genetically engineered strain of Escherichia coli K-12. The system is based on the full-length bacterial luciferase operon coupled to the tetracycline-inducible tetA promoter in the reporter plasmid pTetLux1. Sublethal doses of tetracycline are used to start the luciferase synthesis in cultures that were previously incubated with the antibiotic under investigation. After a variable time frame-from 1 to 4 h, depending on the antimicrobial mode of action-the level of light emission from treated cultures is compared to the level obtained in control cultures. The gap in bioluminescence outlines the antibiotic interference in bacterial metabolism. Throughout this study, freeze-dried sensor cells were used to avoid repeated cultures from day to day. The authors show the results of 10 model antibiotics, representing different molecular structures and mechanisms of action. The results show that no actively dividing cells are needed for sensitive responses, especially when transcriptional and translational inhibitors, directly interfering with the luciferase production, are tested. The assay can be easily automated for high-throughput screening purposes of pharmaceutical industry. (Journal of Biomolecular Screening 2003:340-346)

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“…The resulting RNA hybrid expression vector pRT3'B has numerous advantages over other previously studied induction systems that utilize RNA polymerase II promoters or RNA polymerase III promoters (7)(8)(9). First, the RNA polymerase III-dependent RPR1 promoter has a greater capacity to synthesize RNA transcripts than do RNA polymerase II-dependent promoters.…”

Section: Discussionmentioning

confidence: 99%

“…Previously, the use of human U6 or H1 promoters in the tetracycline-regulatable synthesis of miRNA or shRNA by RNA polymerase III in mammalian systems was reported (3-6) along with tetracycline-regulatable gene expression by RNA polymerase II or III in yeast (7)(8)(9). Furthermore, the tetracycline analogue doxycycline (Dox) has little effect on phenotype or gene expression in yeast, even at high concentrations, when used as an inducing agent in the tetracycline-regulatable promoter system (10).…”

Section: Introductionmentioning

confidence: 99%

On-off controllable RNA hybrid expression vector for yeast three-hybrid system

Bak¹,

Hwang²,

Ko³

et al. 2010

BMB Reports

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The yeast three-hybrid system (Y3H), a powerful method for identifying RNA-binding proteins, still suffers from many false positives, due mostly to RNA-independent interactions. In this study, we attempted to efficiently identify false positives by introducing a tetracycline operator (tetO) motif into the RPR1 promoter of an RNA hybrid expression vector. We successfully developed a tight tetracycline-regulatable RPR1 promoter variant containing a single tetO motif between the transcription start site and the A-box sequence of the RPR1 promoter. Expression from this tetracycline-regulatable RPR1 promoter in the presence of tetracycline-response transcription activator (tTA) was positively controlled by doxycycline (Dox), a derivative of tetracycline. This on-off control runs opposite to the general knowledge that Dox negatively regulates tTA. This positively controlled RPR1 promoter system can therefore efficiently eliminate RNA-independent false positives commonly observed in the Y3H system by directly monitoring RNA hybrid expression. [BMB reports 2010; 43(2): 110-114]

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RNA polymerase III catalysed transcription can be regulated in Saccharomyces cerevisiae by the bacterial tetracycline repressor-operator system.

Cited by 64 publications

References 57 publications

Retrograde movement of tRNAs from the cytoplasm to the nucleus in Saccharomyces cerevisiae

Retrograde movement of tRNAs from the cytoplasm to the nucleus in Saccharomyces cerevisiae

Amplified Detection of Transcriptional and Translational Inhibitors in Bioluminescent Escherichia coli K-12

On-off controllable RNA hybrid expression vector for yeast three-hybrid system

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