A fast and efficient translational control system for conditional expression of yeast genes

Kötter, Peter; Weigand, Julia E.; Meyer, Britta; Entian, Karl‐Dieter; Suess, Beatrix

doi:10.1093/nar/gkp578

Cited by 84 publications

(81 citation statements)

References 31 publications

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“…In this method, one measures, during a chase, the ratio of a test protein to a long-lived reference protein, the mouse dihydrofolate reductase (DHFR). The reference protein and test proteins were coexpressed on a low-copy plasmid from two identical P TDH3 promoters containing additional DNA elements developed by others (53). Once transcribed into an mRNA, these elements form 5′-RNA aptamers that can bind to tetracycline (Tc), thereby repressing translation of that mRNA in cis (Fig.…”

Section: Resultsmentioning

confidence: 99%

Control of Hsp90 chaperone and its clients by N-terminal acetylation and the N-end rule pathway

Hyun

Varshavsky

2017

Proc. Natl. Acad. Sci. U.S.A.

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We found that the heat shock protein 90 (Hsp90) chaperone system of the yeast Saccharomyces cerevisiae is greatly impaired in naa10Δ cells, which lack the NatA N α -terminal acetylase (Nt-acetylase) and therefore cannot N-terminally acetylate a majority of normally N-terminally acetylated proteins, including Hsp90 and most of its cochaperones. Chk1, a mitotic checkpoint kinase and a client of Hsp90, was degraded relatively slowly in wild-type cells but was rapidly destroyed in naa10Δ cells by the Arg/N-end rule pathway, which recognized a C terminus-proximal degron of Chk1. Diverse proteins (in addition to Chk1) that are shown here to be targeted for degradation by the Arg/N-end rule pathway in naa10Δ cells include Kar4, Tup1, Gpd1, Ste11, and also, remarkably, the main Hsp90 chaperone (Hsc82) itself. Protection of Chk1 by Hsp90 could be overridden not only by ablation of the NatA Nt-acetylase but also by overexpression of the Arg/N-end rule pathway in wild-type cells. Split ubiquitin-binding assays detected interactions between Hsp90 and Chk1 in wild-type cells but not in naa10Δ cells. These and related results revealed a major role of Nt-acetylation in the Hsp90-mediated protein homeostasis, a strong up-regulation of the Arg/N-end rule pathway in the absence of NatA, and showed that a number of Hsp90 clients are previously unknown substrates of the Arg/N-end rule pathway.roteins called chaperones promote the in vivo protein folding and counteract misfolding, maladaptive protein aggregation, and other perturbations of proteostasis (1-4). Hsp90 is an ATPdependent chaperone system that mediates the conformational maturation and stabilization of many proteins. Referred to as Hsp90 clients, these proteins include kinases, transcriptional regulators, and many other proteins that comprise at least 20% of a cellular proteome. The diversity of Hsp90 clients and the broad range of processes that involve Hsp90 underlie its necessity for cell viability in all eukaryotes (5-11). Cochaperones of the ∼90-kDa Hsp90 comprise, in mammals, ∼30 distinct proteins. They participate in the delivery of clients to Hsp90 and contribute to related processes, including ATP hydrolysis by Hsp90 (12). Because of its ability to modulate protein conformations, the Hsp90 system can either exacerbate or counteract the fitness-reducing effects of mutations in cellular proteins. This property of Hsp90 affects the standing genetic variation and thereby influences the impact of natural selection on rates and directions of evolution (13,14).The N-end rule pathways comprise a set of proteolytic systems whose unifying feature is their ability to recognize proteins containing N-terminal (Nt) degradation signals called N-degrons, thereby causing the degradation of these proteins by the proteasome or autophagy in eukaryotes and by the proteasome-like ClpAP protease in bacteria (Fig. 1) (15-26). The main determinant of an N-degron is a destabilizing Nt-residue of a protein. Many N-degrons become active through their enzymatic Nt-acetylation, Ntformylation...

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Section: Resultsmentioning

confidence: 99%

Control of Hsp90 chaperone and its clients by N-terminal acetylation and the N-end rule pathway

Hyun

Varshavsky

2017

Proc. Natl. Acad. Sci. U.S.A.

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“…Both aptamers contain two asymmetrical internal loops that are not well ordered in the free RNA, but fold into a compact structure upon ligand binding, with one nucleotide capping the binding pocket. Evidence for a dynamic equilibrium of an ''inactive'' and an ''active'' state was provided for a theophylline-binding RNA aptamer (Latham et al 2009). Their results indicated that the RNA utilize a conformational selection mechanism for binding theophylline like other RNAs with conformationally heterogeneous free states, such as the aptamer domains in RNA riboswitches binding their ligands.…”

Section: Discussionmentioning

confidence: 99%

“…The tetracycline (Tc)-binding aptamer investigated in the present work has been used as an RNA switch for conditional gene expression in yeast, either inhibiting translation initiation (Suess et al 2003;Kötter et al 2009) or pre-mRNA splicing (Weigand and Suess 2007). The ligand Tc inhibits prokaryotic translation (Epe et al 1987;Spahn and Prescott 1996) and is used as a therapeutic agent of low toxicity .…”

Section: Introductionmentioning

confidence: 99%

Ligand-induced conformational capture of a synthetic tetracycline riboswitch revealed by pulse EPR

Wunnicke

Strohbach²,

Weigand³

et al. 2010

RNA

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RNA aptamers are in vitro-selected binding domains that recognize their respective ligand with high affinity and specificity. They are characterized by complex three-dimensional conformations providing preformed binding pockets that undergo conformational changes upon ligand binding. Small molecule-binding aptamers have been exploited as synthetic riboswitches for conditional gene expression in various organisms. In the present study, double electron-electron resonance (DEER) spectroscopy combined with site-directed spin labeling was used to elucidate the conformational transition of a tetracycline aptamer upon ligand binding. Different sites were selected for post-synthetic introduction of either the (1-oxyl-2,2,5,5-tetramethylpyrroline-3-methyl) methanethiosulfonate by reaction with a 4-thiouridine modified RNA or of 4-isocyanato-2,6-tetramethylpiperidyl-N-oxid spin label by reaction with 29-aminouridine modified RNA. The results of the DEER experiments indicate the presence of a thermodynamic equilibrium between two aptamer conformations in the free state and capture of one conformation upon tetracycline binding.

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“…Furthermore, the reporter gene in use seems to have an impact on the result as well. Fowler et al 40 and K€ otter et al 41 observed that the insertion of secondary structure elements in the 5 0 -UTR of a GFP gene can lead to a reduction in gene expression and, consequently, fluorescence intensity. Obviously, some of the riboswitch elements provoke a similar reduction.…”

Section: Increasing the Activation Ratio By Tandem And Tridem Constructsmentioning

confidence: 99%

Design criteria for synthetic riboswitches acting on transcription

et al. 2015

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Riboswitches are RNA-based regulators of gene expression composed of a ligand-sensing aptamer domain followed by an overlapping expression platform. The regulation occurs at either the level of transcription (by formation of terminator or antiterminator structures) or translation (by presentation or sequestering of the ribosomal binding site). Due to a modular composition, these elements can be manipulated by combining different aptamers and expression platforms and therefore represent useful tools to regulate gene expression in synthetic biology. Using computationally designed theophylline-dependent riboswitches we show that 2 parameters, terminator hairpin stability and folding traps, have a major impact on the functionality of the designed constructs. These have to be considered very carefully during design phase. Furthermore, a combination of several copies of individual riboswitches leads to a much improved activation ratio between induced and uninduced gene activity and to a linear dose-dependent increase in reporter gene expression. Such serial arrangements of synthetic riboswitches closely resemble their natural counterparts and may form the basis for simple quantitative read out systems for the detection of specific target molecules in the cell.

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A fast and efficient translational control system for conditional expression of yeast genes

Cited by 84 publications

References 31 publications

Control of Hsp90 chaperone and its clients by N-terminal acetylation and the N-end rule pathway

Control of Hsp90 chaperone and its clients by N-terminal acetylation and the N-end rule pathway

Ligand-induced conformational capture of a synthetic tetracycline riboswitch revealed by pulse EPR

Design criteria for synthetic riboswitches acting on transcription

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