Context Effects on Misreading and Suppression at UAG Codons in Human Cells

Abstract

The effect of the 3 codon context on the efficiency of nonsense suppression in mammalian tissue culture cells has been tested. Measurements were made following the transfection of cells with a pRSVgal reporter vector that contained the classical Escherichia coli lacZ UAG allele YA559. The position of this mutation was mapped by virtue of its fortuitous creation of a CTAG MaeI restriction enzyme site. Determination of the local DNA sequence revealed a C3T mutation at codon 600 of the lacZ gene: CAG3TAG. Site-directed mutagenesis was used to create a series of vectors in which the base 3 to the nonsense codon was either A, C, G, or U. Suppression of the amber-containing reporter was achieved by cotransfection with genes for human tRNA Ser or tRNA Gln UAG nonsense suppressors and by growth in the translational error-promoting aminoglycoside drug G418. Nonsense suppression was studied in the human cell lines 293 and MRC5V1 and the simian line COS-7. Overall, the rank order for the effect of changes to the base 3 to UAG was C < G ‫؍‬ U < A. This study confirms and extends earlier findings that in mammalian cells 3 C supports efficient nonsense suppression while 3 A is unsympathetic for read-through at nonsense codons. The rules for the mammalian codon context effect on nonsense suppression are therefore demonstrably different from those in E. coli.Normally, nonsense or stop codons catalyze the effective cessation of protein synthesis. However, when tRNA species within the cell are able to recognize a nonsense codon, the function of the stop is suppressed. Nonsense suppression has been exploited as a means for the conditional expression of genes containing stop codons in order to study their function (23,44). Nonsense suppression has also been used extensively in Escherichia coli as a tool for monitoring the efficiency of translation at an individual codon (46,47,51). Of particular interest is the facility that nonsense suppression affords for determining the effects of 5Ј and 3Ј contexts on the interaction of translation factors and aminoacyl-tRNAs with their mRNA targets (19,39,45,49,52). Recently, nonsense suppression has been harnessed in order to carry out in vitro protein engineering. In this process, chemically acylated nonsense suppressor tRNAs are used to insert nonnatural amino acid residues at specified locations by targeting a nonsense codon which interrupts the coding sequence (2, 3, 33).There have been comparatively few reports of nonsense suppression in mammalian cells, but the transfection of sensitively detected reporters now allows this technique to be applied to cells in culture (6,12,13,15,43). In a previous report from this laboratory, we described the effects of alterations in the 3Ј mRNA context on the efficiency of a human tRNA Ser UAG nonsense suppressor (40). The function of the suppressor was monitored by determining the efficiency of translation at a UAG codon in a ␤-galactosidase reporter gene which had been cotransfected into human 293 cells with a plasmid vector expressing a human UAG su...

L-MPZ, a Novel Isoform of Myelin P0, Is Produced by Stop Codon Readthrough

“…6A and 9A). In addition, nonsense suppression using suppressor tRNA is enhanced by G418 in mammalian culture cells (31). Because L-MPZ synthesis with in vitro transcription/translation was dramatically increased by G418, L-MPZ may be produced by the suppression of the regular P0 stop codon.…”

A temperature-sensitive mutation in thenodal-related genecyclopsreveals that the floor plate is induced during gastrulation in zebrafish

“…Because an alternate start site (met 336) is present within the pro region after the stop codon, it is possible that the N terminus-truncated Cyc protein generated from this site is stable and functional in zebrafish embryos at 22°C but not at 28.5°C. Alternatively, a translational read-though mechanism similar to that described in mammalian cells (Laski et al, 1982;Hryniewicz and Vonder Haar, 1983;Phillips-Jones et al, 1995) may function at 22°C, the permissive temperature for cyc sg1 , allowing Cyc function at this temperature. It is also possible that the pro domain of Cyc has some activities that have not been previously identified.…”

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

“…The efficiency of termination and that of nonsense suppression are influenced by the 3Ј codon context, with termination ruled by the base immediately following the termination codon (34). In particular, the efficiency of suppression of an amber codon in human tissue culture cells varies according to the 3Ј base in the pattern C Ͻ G ϭ U Ͻ A (41). In addition, the physical and chemical characteristics of the last two amino acids in the nascent peptide function as additional codon context determinants in both bacteria and Saccharomyces cerevisiae (7,8,32).…”