Charakterisierung der Ubiquitinierung des Ku80-Proteins

Die an das humane Ku80-Protein angehefteten Ubiquitinketten sind hauptsächlich über die Lysine an den Positionen 33 und 63 miteinander verbunden.Eine Überexpression von BRCA1 bzw. RNF8 führt zu einer verstärkten Ubiquitinierung des humanen Ku80-Proteins.In einem Ku80-Fragment mit 168 Aminosäuren sind die Lysine an den Positionen 125/126/129 für die Ubiquitinierung verantwortlich.Die Reduktion der BRCA1 Menge mittels siRNA führt zu einer verringerten Bindung von humanem Ku80 an den DSB

RsfA (YbeB) Proteins Are Conserved Ribosomal Silencing Factors

The YbeB (DUF143) family of uncharacterized proteins is encoded by almost all bacterial and eukaryotic genomes but not archaea. While they have been shown to be associated with ribosomes, their molecular function remains unclear. Here we show that YbeB is a ribosomal silencing factor (RsfA) in the stationary growth phase and during the transition from rich to poor media. A knock-out of the rsfA gene shows two strong phenotypes: (i) the viability of the mutant cells are sharply impaired during stationary phase (as shown by viability competition assays), and (ii) during transition from rich to poor media the mutant cells adapt slowly and show a growth block of more than 10 hours (as shown by growth competition assays). RsfA silences translation by binding to the L14 protein of the large ribosomal subunit and, as a consequence, impairs subunit joining (as shown by molecular modeling, reporter gene analysis, in vitro translation assays, and sucrose gradient analysis). This particular interaction is conserved in all species tested, including Escherichia coli, Treponema pallidum, Streptococcus pneumoniae, Synechocystis PCC 6803, as well as human mitochondria and maize chloroplasts (as demonstrated by yeast two-hybrid tests, pull-downs, and mutagenesis). RsfA is unrelated to the eukaryotic ribosomal anti-association/60S-assembly factor eIF6, which also binds to L14, and is the first such factor in bacteria and organelles. RsfA helps cells to adapt to slow-growth/stationary phase conditions by down-regulating protein synthesis, one of the most energy-consuming processes in both bacterial and eukaryotic cells

RsfA and L14 and their interaction are conserved in bacteria and eukaryotic organelles.

<p>(A) Phylogenetic distribution of RsfA (Interpro entry IPR004394 [DUF143]) and ribosomal protein L14 (IPR000218) on the iTOL tree of life <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002815#pgen.1002815-Letunic1" target="_blank">[59]</a>. Triangles indicate species in which the RsfA-L14 interaction was detected by binary detection assays (grey), co-purification with the LRS (white) or both (black). Known RsfA-L14/LRS interactions are listed in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002815#pgen.1002815.s005" target="_blank">Table S1</a>. (B) <i>T. pallidum</i> RsfA (TP0738) interacts strongly with L14 (TP0199) and very weakly with other proteins involved in translation <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002815#pgen.1002815-Titz1" target="_blank">[6]</a> in yeast-two-hybrid assays. C, control (with empty prey vector to measure self-activation of the bait). This interaction is also conserved in <i>E. coli</i> (C). (D, E) RsfA and L14 homologues from human and maize interact in pull down experiments. RsfA homologues were tagged with NusA-His₆ (N) and L14 homologues with maltose binding protein (M) (human mtRsfA = C7orf30, mitochondrial ribosomal protein L14 = L14_mt; maize RsfA = Iojap, maize chloroplastic L14 = RPL14); i = input samples, o = output samples. Constructs with the corresponding Interpro signatures and the range of cloned codons are illustrated on the right. (F) Human mitochondrial C7orf30 (mtRsfA) co-localizes with L14_mt exclusively into mitochondria as visualized by MitoTracker Green. Nuclei visualized by DRAQ5 (blue) and membranes by eCFP-membrane (cyan). Co-localization of both mtRsfA (C7orf30) and L14_mt in mitochondria is indicated in yellow. (G) Bi-molecular fluorescence complementation (BiFC) reveals the interaction of mtRsfA (C7orf30) and L14_mt in mitochondria. Overlay images represent DRAQ5 (blue), CFP-membrane (cyan) and BiFC stained cells. Green fluorescence indicates interaction-dependent regeneration of the Venus protein. Constructs are shown below. Here, the hexagons symbolize the native N-termini including mitochondrial localization sequences.</p

A model of RsfA action.

<p>In rich medium and during exponential growth, RsfA is either not present or not active, so that protein synthesis is fully active. In starving cells, RsfA binds to ribosomal L14 and, as a consequence, blocks ribosomal subunit joining and thus protein synthesis.</p

Mapping the RsfA binding site on ribosomal protein L14.

<p>(A) L14 in the context of the 3D structure of the 50S ribosomal subunit (a) (PDB: 2AWB) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002815#pgen.1002815-Schuwirth1" target="_blank">[14]</a>. (b) Conserved residues of L14: magenta (highly conserved), grey (moderately conserved), turquoise (little or no conservation). (c) Mutated residues for interaction epitope mapping (red or green); residues involved in (red colors) and not involved (green colors) in RsfA-binding based on results from subfigure (C). (d) Residues of L14 highlighted that are involved in formation of intersubunit bridges with the 16S rRNA of the 30S subunit (bridge B5 (green colors), bridge B8 (red colors)) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002815#pgen.1002815-Gao1" target="_blank">[15]</a>. (B) A docking model of L14 on the <i>E. coli</i> 50S subunit with bound RsfA. Critical L14 residues that mediate RsfA interaction (or that contact 16S rRNA) are colored in red according to A(c) and A(d). When RsfA is bound to L14 on a 50S subunit, 30S subunit joining is sterically blocked, clearly visible in B(b) as shown by the structural overlap of RsfA (dark blue) and the 30S subunit. A model of the ribosome with bound RsfA is available as <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002815#pgen.1002815.s001" target="_blank">Dataset S1</a>. (C) L14 interaction epitope mapping. Amino acids (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002815#pgen-1002815-g002" target="_blank">Figure 2A(c)</a>) were mutated to alanine and the constructs tested by Y2H experiments. WT, wild type L14 construct; mutated residues and their positions are indicated. In the experiment, all bait constructs were simultaneously tested for reporter gene self-activation. No construct resulted in self-activation (data not shown). T97A, R98A, or K114A mutations (highlighted by arrows) abolished or weakened RsfA binding as indicated by 3-AT titrations; all other tested L14 mutation constructs are comparable to wild type L14.</p

RsfA inhibits translation by blocking ribosomal subunit joining.

<p>(A) Oligo(Phe) synthesis in a pure system containing pre-charged Phe-tRNAs (ten times over ribosomes), 30S and 50S subunits and the purified factors EF-Tu, EF-Ts and EF-G plus/minus RsfA from <i>E. coli</i>, 100% corresponds to 7 Phe incorporated per ribosome. Left panel, when indicated RsfA was added to the 50S subunits, before 30S subunits were added starting oligo(Phe) synthesis. Right panel, AcPhe-tRNA was bound to 70S ribosomes in the presence of poly(U) before the addition of RsfA. (B) Sister-aliquots from the same samples shown in (A) were analyzed on a sucrose gradient before oligo(Phe) synthesis. The presence of RsfA significantly reduces the fraction of 70S ribosomes. (C) Oligo(Phe)-synthesis as in (A) but with purified mitochondrial components (pig liver) and human mtRsfA (C7orf30). 39S and 28S indicate the large and small ribosomal subunits, 55S the associated mitochondrial ribosomes. For details see Experimental Procedures.</p

RsfA inhibits translation during both stationary phase and the transition from rich to poor media.

<p>(A) Growth competition experiment: equal numbers of <i>E. coli</i> wild type and <i>ΔrsfA</i> cells derived from an overnight LB-culture were mixed and grown in rich medium (LB, rich→rich), poor medium (M9, rich→poor) and poor medium plus 2% casamino acids as indicated (rich→poor+aa). Growth was maintained in log phase conditions by regular dilutions in the corresponding media. Shown is the fraction of viable <i>ΔrsfA</i> mutant cells in the total cell population. (B) Wild type and mutant strains were grown overnight in rich medium (LB) and then diluted in rich (rich→rich) or poor M9 medium (rich→poor). The generation time was derived from the slopes of the regression lines made of the points indicating the logarithmic phase. The errors of the generation-time determinations are below ±5%, <i>i.e.</i> generation times of 30 and 32 min are not significantly different. (C) Wild type and mutant strains transformed with a plasmid harboring the gene for RsfA fused with a His-tag under control of the native promoter or the corresponding empty plasmid were grown overnight in rich medium (LB) and then diluted in poor M9 medium. At certain times samples were withdrawn (S1–S6) and the relative amount of RsfA was quantified by Western-blot (represented with bars). S1–S3: samples were analyzed from both strains. S4–S6: samples were analyzed only from wild type (blue) or mutant strain (red). (D) Same as (C) but using a plasmid with a His-tagged RsfA gene under a tac promoter. After ∼3 h incubation in M9 medium 0.2 mM IPTG (final concentration) was added to all strains in order to induce expression from the tac promoter. (E) Viability competition similar to the growth competition described under (A) but in a batch culture without dilution. Red, growth of the mixture of <i>ΔrsfA</i> and WT strains; blue, the fraction (in %) of the mutant strain. (F) Expression of β-galactosidase as reporter to test translational activity of logarithmic and stationary phase cells in WT and <i>ΔrsfA</i> cells induced by 2% arabinose. Induction time was 3 h in logarithmic and 2.5 and 6.5 h in stationary phase. The expression level was derived from the band-intensity on a gel (Coomassie-stained SDS-PAGE).</p