Establishment of a system for conditional gene expression using an inducible tRNA suppressor gene.

Abstract

We investigated the use of the prokaryotic tetracycline operator-repressor system as a regulatory device to control the expression of Dictyostelium discoideum tRNA genes. The tetO_ operator fragment was inserted at three different positions in front of a tRNAGrU(Am) suppressor gene from D. discoideum, and the tetracycline repressor gene was expressed under the control of a constitutive actin 6 promoter. The effectiveness of this approach was determined by monitoring the expression of a 1-galactosidase gene engineered to contain a stop codon that could be suppressed by the tRNA. When these constructs were introduced into Dictyostelium cells, the repressor bound to the operator in front of the tRNA gene and prevented expression of the suppressor tRNA. Addition of tetracycline (30 ,g/ml) to the growth medium prevented repressor binding, allowed expression of the suppressor tRNA, and resulted in f-galactosidase synthesis. The operator-repressor complex interfered with tRNA gene transcription when the operator was inserted immediately upstream (position +1 or -7) of the mature tRNA coding region. Expression of a tRNA gene carrying the operator at position -46 did not respond to repressor binding. This system could be used to control the synthesis of any protein, provided the gene contained a translational stop signal.tRNA suppressors are a classic means of regulating the expression of a protein. In the absence of a suppressor tRNA, the mRNA derived from a gene which has been mutated to contain a stop codon will be incompletely translated. When a tRNA suppressor gene is introduced into the cell, an authentic protein is synthesized, provided the suppressor tRNA inserts the same amino acid as the cognate tRNA of the unmutated codon. This is a powerful tool for analysis of the function of any gene but has been relegated primarily to procaryotes because of the difficulty of manipulating tRNA expression in eukaryotic cells. Eukaryotic tRNA genes contain gene-internal polymerase III (polIII) promoters (for reviews, see references 23 and 46). These transcriptional control regions become part of the mature tRNA coding sequence and are indispensable for tRNA function. As a consequence, these regions are difficult to manipulate. Another consequence is that other than in the case of polII genes, polIII gene promoters cannot be replaced by regulated polII promoters, such as the heat shock and metallothionein promoters.In addition to the gene-internal control elements, 5'-flanking regions of tRNA genes frequently exert a modulatory influence on gene expression (1-3, 10, 15, 17, 27, 37, 41, 47, 49). Although the mechanism of tRNA gene modulation by 5'-flanking regions is not understood, we recently demonstrated that stable binding of a protein near a tRNA gene strongly inhibits its expression (39). This inhibition apparently resulted from masking of the initiation site for tRNA gene transcription by the bound protein and from a strong interaction of the protein with the RNA polIII transcription complex. These findings suggested th...

Tetracycline-Regulated Suppression of Amber Codons in Mammalian Cells

“…This level of regulation is similar to that seen in other transienttransfection systems (1,21,43,45) or in D. discoideum (10), where tetracycline is used to regulate expression of the suppressor tRNA rather than its function. The 15-to 78-fold regulation of amber suppression by tetracycline in HeLa cells is, however, substantially less than that in a stable HeLa cell line expressing the same tTA constitutively (17) or in animals using different transactivation systems induced by mifepristone (RU486) or ecdysone (31,43,44).…”

“…Other attempts to regulate suppressor tRNA gene expression have used the E. coli lac repressor to block the expression of a suppressor tRNA gene linked on the 5Ј side to a lac operator sequence (39,41). Similarly, the tetracycline repressor-operator system has been used to regulate the expression of a Dictyostelium discoideum amber suppressor tRNA gene in D. discoideum and in the yeast Saccharomyces cerevisiae (9,10).…”

Silkworm TFIIIB Binds both Constitutive and Silk Gland-Specific tRNAAla Promoters but Protects Only the Constitutive Promoter from DNase I Cleavage

“…(i) Template commitment assays show that TFIIIB is stably sequestered by preformed TFIIIC-template complexes (1,45). (ii) Transcription is severely impaired if exotic proteins are permitted to bind within upstream regions corresponding to the binding site of yeast TFIIIB (9,28,31,54). The principal difficulty in detecting TFIIIB-DNA complexes directly may be due to low concentrations of the required factors.…”

Suppression of Nonsense Mutations in Cell Culture and Mice by Multimerized Suppressor tRNA Genes

“…These approaches induced repression or activation of constitutive suppressor tRNA expression (16,49,52) as well as control of suppressor tRNA function by modulation of the aminoacylation process (18,39).…”