Saccharomyces cerevisiae protein phosphorylation and translation accuracy
Data files
Jan 23, 2024 version files 26.04 KB
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Effect_Ctk1_dependent_phosphorylation_on_accuracy.xlsx
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README.md
Abstract
Protein-protein and protein-rRNA interactions involving ribosomal proteins uS4 and uS5 are thought to maintain the accuracy of protein synthesis by increasing selection of cognate aminoacyl-tRNAs. Selectivity involves a major conformational change—domain closure—that stabilizes aminoacyl-tRNA in the ribosomal acceptor (A) site. This has been thought a constitutive function of the ribosome ensuring consistent accuracy. Recently, the Saccharomyces cerevisiae Ctk1 cyclin-dependent kinase was demonstrated to ensure translational accuracy and Ser238 of uS5 proposed as its target. Surprisingly, Ser238 is outside the uS4-uS5 interface and no obvious mechanism has been proposed to explain its role. We show that the true target of Ctk1 regulation is another uS5 residue, Ser176, which lies in the interface opposite to Arg57 of uS4. Based on site-specific mutagenesis, we propose that phospho-Ser176 forms a salt bridge with Arg57, which should increase selectivity by strengthening the interface. Genetic data show that Ctk1 regulates accuracy indirectly by stimulating phosphorylation of Ser176 by the kinase Ypk2. A second kinase pathway involving TORC1 and Pkc1 can inhibit this effect. The level of accuracy appears to depend on competitive action of these two pathways to regulate the level of Ser176 phosphorylation.
README: Protein phosphorylation and translation accuracy
https://doi.org/10.5061/dryad.jdfn2z3gs
The data consist of the results of in vivo misreading reporter assays. The reporter consists of the Escherichia coli lacZ gene, encoding beta-galactosidase, expressed in the yeast Saccharomyces cerevisiae from an autonomously replicating plasmid. The data come from the Beta-Glo system in which galactosyl-luciferin is cleaved by beta-galactosidase releasing luciferin, which is the substrate for a light-dependent reaction with firefly luciferase. The assay is a one-hour timed reaction of excess galactosyl-luciferin with the beta-galactosidase produced in vivo. The amount of luciferin released is then determined in a timed reaction in the presence of excess luciferase. The relative light units (RLUs) produced in this reaction are proportional to misreading frequency and is quantitated by calculating the ratio of RLU produced using extract of a misreading reporter mutant beta-galactosidase enzyme divided by the RLU produced from a wild-type beta-galactosidase enzyme. The ratio is equal to the frequency of misreading in vivo for the mutant beta-galactosidase gene.
Description of the data and file structure
The data presented is the ratio of beta-galactosidase activity in the mutant to the wild-type control. Each number is the average of three technical repeats (three assays of the same protein extract). Each row of the data file includes a reference to the identity of the genetic background of the cells used to produce the protein extract, the near-cognate codon misread by tRNA(Glu), and from three to twenty-four independent, biological repeats of the assay for that genetic background.
The data come in sections that are identified at the beginning by a description of the purpose of each experiment.
Sharing/Access information
This data is only available through Dryad
Methods
This dataset is the result of in vivo analysis of misreading error frequency in the yeast Saccharomyces cerevisiae using a set of misreading error reporter systems developed in our laboratory.