An in vivo dual-luciferase assay system for studying translational recoding in the yeast <i>Saccharomyces cerevisiae</i>

Harger, Jason W.; Dinman, Jonathan D.

doi:10.1261/rna.5930803

Cited by 155 publications

(222 citation statements)

References 22 publications

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“…Dual luciferase assays were performed with reporter systems to measure nonsense and missense suppression. Nonsense suppression assays were performed on strains containing URA3-based CEN plasmids containing renilla and firefly luciferase reporters expressed from the ADH1 promoter with the CYC1 terminator and either the wild-type sequence (AAA) or a stop codon (UAA) (Harger and Dinman 2003). Missense suppression assays were performed on strains containing URA3-based CEN plasmids containing a CAC (His)-to-CGC (Arg) mutation in firefly luciferase at codon 245 (Salas-Marco and Bedwell 2005).…”

Section: Methodsmentioning

confidence: 99%

Unique Classes of Mutations in the Saccharomyces cerevisiae G-Protein Translation Elongation Factor 1A Suppress the Requirement for Guanine Nucleotide Exchange

et al. 2006

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G-proteins play critical roles in many cellular processes and are regulated by accessory proteins that modulate the nucleotide-bound state. Such proteins, including eukaryotic translation elongation factor 1A (eEF1A), are frequently reactivated by guanine nucleotide exchange factors (GEFs). In the yeast Saccharomyces cerevisiae, only the catalytic subunit of the GEF complex, eEF1Ba, is essential for viability. The requirement for the TEF5 gene encoding eEF1Ba can be suppressed by the presence of excess substrate, eEF1A. These cells, however, have defects in growth and translation. Two independent unbiased screens performed to dissect the cause of these phenotypes yielded dominant suppressors that bypass the requirement for extra eEF1A. Surprisingly, all mutations are in the G-protein eEF1A and cluster in its GTP-binding domain. Five mutants were used to construct novel strains expressing only the eEF1A mutant at normal levels. These strains show no growth defects and little to no decreases in total translation, which raises questions as to the evolutionary expression of GEF complexity and other potential functions of this complex. The location of the mutations on the eEF1A-eEF1Ba structure suggests that their mechanism of suppression may depend on effects on the conserved G-protein elements: the P-loop and NKXD nucleotide-binding element.

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Section: Methodsmentioning

confidence: 99%

Unique Classes of Mutations in the Saccharomyces cerevisiae G-Protein Translation Elongation Factor 1A Suppress the Requirement for Guanine Nucleotide Exchange

et al. 2006

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“…Subsequently, 10 4 CFU (5 μl) aliquots, and tenfold dilutions from the same cultures thereof were spotted either onto rich medium and incubated at 15, 30 and 37°C, or incubated at 30°C on rich medium containing anisomycin (10 μg/ml) or sparsomycin (30 μg/ml). Assays for programmed ribosomal frameshifting followed previously described protocols (Smith et al 2001;Harger and Dinman 2003). These involve the use of 0-frame control and −1 or +1 ribosomal frameshift test vectors in which the production of a reporter enzyme (either firefly luciferase of β-galactosidase) is dependent upon a programmed ribosomal frameshift event.…”

Section: Strains and Genetic Methodsmentioning

confidence: 99%

Structural and functional analysis of 5S rRNA in Saccharomyces cerevisiae

et al. 2005

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Abstract5S rRNA extends from the central protuberance of the large ribosomal subunit, through the A-site finger, and down to the GTPase-associated center. Here, we present a structure-function analysis of seven 5S rRNA alleles which are sufficient for viability in the yeast Saccharomyces cerevisiae when expressed in the absence of wild-type 5S rRNAs, and extend this analysis using a large bank of mutant alleles that show semidominant phenotypes in the presence of wild-type 5S rRNA. This analysis supports the hypothesis that 5S rRNA serves to link together several different functional centers of the ribosome. Data are also presented which suggest that in eukaryotic genomes selection has favored the maintenance of multiple alleles of 5S rRNA, and that these may provide cells with a mechanism to post-transcriptionally regulate gene expression.

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“…32 To analyze the involvement of uL11 in the PRF, we used a reporter system designed for quantification of ribosomal frameshifting directly in vivo. 33 The system applies dual-luciferase reporters harboring a specific yeast L-A viral -1 PRF signal and a Ty1 C1 PRF signal, respectively. The results presented in Figure 3B show that ribosomes deprived of uL11 proteins exhibit a significant ORF maintenance defect.…”

Section: Translation Accuracymentioning

confidence: 99%

“…33 Briefly, each yeast strain was transformed with a control plasmid pYDL and with an appropriate reporter plasmid for -1 PRF (pYDL-LA) and C1 PRF (pYDL-Ty1), respectively. Transformants were grown on SD minimal medium without Uracil to OD 600 0.5-0.7.…”

Section: Rna Electrophoresis and Northern Hybridizationmentioning

confidence: 99%

Functional analysis of the uL11 protein impact on translational machinery

Wawiórka¹,

Molestak²,

Szajwaj³

et al. 2016

Cell Cycle

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The ribosomal GTPase associated center constitutes the ribosomal area, which is the landing platform for translational GTPases and stimulates their hydrolytic activity. The ribosomal stalk represents a landmark structure in this center, and in eukaryotes is composed of uL11, uL10 and P1/P2 proteins. The modus operandi of the uL11 protein has not been exhaustively studied in vivo neither in prokaryotic nor in eukaryotic cells. Using a yeast model, we have brought functional insight into the translational apparatus deprived of uL11, filling the gap between structural and biochemical studies. We show that the uL11 is an important element in various aspects of 'ribosomal life'. uL11 is involved in 'birth' (biogenesis and initiation), by taking part in Tif6 release and contributing to ribosomal subunit-joining at the initiation step of translation. uL11 is particularly engaged in the 'active life' of the ribosome, in elongation, being responsible for the interplay with eEF1A and fidelity of translation and contributing to a lesser extent to eEF2-dependent translocation. Our results define the uL11 protein as a critical GAC element universally involved in trGTPase 'productive state' stabilization, being primarily a part of the ribosomal element allosterically contributing to the fidelity of the decoding event.

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An in vivo dual-luciferase assay system for studying translational recoding in the yeast Saccharomyces cerevisiae

Cited by 155 publications

References 22 publications

Unique Classes of Mutations in the Saccharomyces cerevisiae G-Protein Translation Elongation Factor 1A Suppress the Requirement for Guanine Nucleotide Exchange

Unique Classes of Mutations in the Saccharomyces cerevisiae G-Protein Translation Elongation Factor 1A Suppress the Requirement for Guanine Nucleotide Exchange

Structural and functional analysis of 5S rRNA in Saccharomyces cerevisiae

Functional analysis of the uL11 protein impact on translational machinery

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