DOI: 10.5061/dryad.jdfn2z3gs
title: Saccharomyces cerevisiae protein phosphorylation and translation accuracy

description: 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.
description: This dataset is the result of in vivo analysis of misreading
 error frequency in the yeast <em>Saccharomyces</em>
 <em>cerevisiae</em> using a set of misreading error reporter
 systems developed in our laboratory.
description: # Protein phosphorylation and translation accuracy
 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