10 research outputs found
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
RsfS (YbeB) reguliert in allen Bakterien und Organellen die ribosomale TranslationsaktivitÀt
Wenn sich Bakterien der stationÀren Phase nÀhern, reduziert sich ihre
Teilungsrate, die metabolischen AktivitĂ€ten werden zurĂŒck gefahren und die
TranslationsaktivitÀt der Ribosomen nimmt rapide ab. Letzteres bedingt den
wohlbekannten Effekt, daà Ribosomen, isoliert von Zellen aus der stationÀren
Phase, nur eine geringe AktivitÀt in vitro haben. Wir haben nur geringe
Kenntnis von den Ursachen der reduzierten ribosomalen AktivitĂ€t (âsilencingâ)
wÀhrend der stationÀren Phase und unter Stressbedingungen. Faktoren wie RMF,
HPF und sein Homolog PY wurden vorgeschlagen, ĂŒber Dimerisation von 70S
inaktive 100S Partikel zu bilden. Jedoch gibt es keine Ăbereinstimmung ĂŒber
die Rolle und das Auftauchen dieser Partikel. Ferner ist bekannt, daĂ die
Entfernung des RMF Gens die LebensfÀhigkeit von E. coli Zellen in der
stationÀren Phase verschlechtert. Weiterhin wurden 100S Partikel auch in
logarithmischer Phase beobachtet, was heiĂen mag, daĂ die 100S Partikel eine
andere Rolle spielt oder weitere Funktionen besitzt. Wir prÀsentieren hier
eine Studie des kĂŒrzlich von uns und mit meiner Beteiligung beschriebenen
âRibosome Silencing Factorâ (RsfS, frĂŒherer Name YbeB), ein Protein das mit
Ribosomen assoziiert ist. RsfS kommt fast in allen Bakterien, Mitochondrien
und Chloroplasten vor und bindet an L14 der groĂen, ribosomalen Untereinheit,
eines der am besten konservierten Proteine des Ribosoms. Die Wechselwirkung
von RsfS mit L14 ist vom Bakterien bis zum Menschen konserviert. Wir zeigen,
daĂ RsfS wichtig fĂŒr das Ăberleben ist, wenn immer die Wachstumsrate herunter
gefahren werden muĂ, d.h. wĂ€hrend des Ăbergangs von der logarithmischen zur
stationÀren Phase oder vom reichen zum armen Medium. Im letzteren Fall ist das
Wachstum blockiert, bis es nach etwa 15 h langsam wieder Fahrt aufnimmt.
Entfernung des RsfS Gens erhöht die TranslationsaktivitÀt in der stationÀren
aber nicht in der logarithmischen Phase. In vitro hemmt RsfS und sein
mitochondriales Homolog die Translation ĂŒber die Bindung an L14 in der groĂen
ribosomalen Untereinheit und blockiert damit die 70S Bildung aus
Untereinheiten oder dissoziiert leere 70S Ribosomen. Interessanterweise wird
die Effizienz zur Dissoziation empfindlich gestört, wenn programmierte
Ribosomen tRNAs tragen. RsfS hemmt nicht die Translation von chemisch
vernetzten 70S Ribosomen, woraus wir schlieĂen, daĂ die Translationshemmung
zum wesentliche Teil auf Dissoziation der 70S Ribosomen bzw. Hemmung der
Assoziation der ribosomalen Untereinheiten zurĂŒckgefĂŒhrt werden kann. Wir
haben auch RsfS mit den Faktoren RMF, PY und HPF genetisch und funktionell
verglichen, um die jeweilige Bedeutung und eine mögliche Kooperation zu
entdecken. Wir fanden, (i) daĂ 100S Bildung keine obligates Merkmal der
stationĂ€ren Phase ist, (ii) daĂ der schwere PhĂ€notyp des ÎrsfS Stammes mit
entsprechenden KO-Mutanten der drei anderen Faktoren nicht beobachtet wird,
(iii) daĂ die LebensfĂ€higkeit von ÎrsfS Zellen und besonders der Îrmf Zellen,
aber nicht der Îhpf or Îpy stark eingeschrĂ€nkt ist, und daĂ schlieĂlich in
vitro RsfS die stĂ€rkste Translationshemmung sowohl bei natĂŒrlichen mRNAs als
auch bei hoch definierten Elongationsexperimenten zeigt. Die Hemmung der
anderen Faktoren ist additiv, nicht kooperativ. ZusammengefaĂt zeigen unsere
Daten, daĂ RsfS eine SchlĂŒsselrolle fĂŒr das ribosomale âsilencingâ hat, wobei
es von den anderen Faktoren unterstĂŒtzt wird.Bacterial cells approaching stationary growth phase reduce division rates, cut
back metabolic activities and thus decrease protein translation causing the
well-known effect that ribosomes isolated from stationary growth phase show a
low translational activity. We have a scarce knowledge about the mechanism of
ribosome silencing during stationary growth phase or under stress condition.
HPF, its homolog PY and RMF have been proposed to bring translation to a halt
by dimerization of 70S ribosomes into 100S particles. However, there is no
consensus about the function or occurrence of 100S particles. Deletion of RMF
decreases the viability of E. coli in stationary growth phase. On the other
hand, 100S particles have been observed also in logarithmic growth phase,
suggesting that 100S particles have a different role than ribosome silencing.
Here we present a study of the recently (with my participation) described
Ribosome Silencing Factor S (former name YbeB), a protein associated with
ribosomes. RsfS is present in almost all bacteria, mitochondria and
chloroplasts and binds to protein L14, one of the most conserved proteins of
the large ribosomal subunit. This interaction is conserved from bacteria to
man. We demonstrate that RsfS is important for cell survival, whenever the
growth rate has to be decreased, i.e. during the transition from the
logarithmic growth phase to the stationary growth phase and from rich to poor
media. In the latter case ÎrsfS strain stops growing for about 15 h before
growth is launched again. Deletion of RsfS gene increases the translation
activity during stationary growth phase but not during logarithmic growth
phase. In vitro RsfS and its mitochondrial homolog inhibits translation by
binding to 50S protein L14 and thus i) inhibits 70S formation from subunits
and ii) dissociates empty 70S ribosomes. Interestingly, the efficiency of 70S
dissociation decreases, when 70S is programmed with tRNA at the P site. RsfS
does not inhibit translation of non dissociable 70S suggesting that ribosome
silencing by RsfS is mediated predominantly via 70S dissociation or anti-
association of the ribosomal subunits. We also compared RsfS with RMF, PY and
HPF to elucidate the importance and possible interplay between these factors
in ribosome silencing. We found that i) 100S formation is not an obligatory
feature of stationary-phase E. coli cells; ii) the strong phenotype of ÎrsfS
strain, viz. a block of growth for about 15 h after transfer from rich to poor
media, is not seen with the knock-out of one of the other factors; (iii)
viability at stationary phase is impaired in ÎrsfS cells and even stronger in
Îrmf cells, but not in Îhpf or Îpy cells; (iv) RsfS is the only factor that
impairs translation in stationary growth phase; (v) in vitro RsfS shows the
strongest inhibition in both translation of natural mRNAs and in highly
resolved elongation assays; the inhibition of the other factors is additive,
not cooperative. Collectively, the data indicate that RsfS plays a key role in
silencing the ribosomal activity under conditions characterized by a reduced
growth rate, and that it is supported by the other silencing factors
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
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-His<sub>6</sub> (N) and L14 homologues with maltose binding protein (M) (human mtRsfAâ=âC7orf30, mitochondrial ribosomal protein L14â=âL14<sub>mt</sub>; maize RsfAâ=âIojap, maize chloroplastic L14â=âRPL14); iâ=âinput samples, oâ=âoutput 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<sub>mt</sub> 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<sub>mt</sub> in mitochondria is indicated in yellow. (G) Bi-molecular fluorescence complementation (BiFC) reveals the interaction of mtRsfA (C7orf30) and L14<sub>mt</sub> 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
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>ÎrsfA</i> cells derived from an overnight LB-culture were mixed and grown in rich medium (LB, richârich), poor medium (M9, richâpoor) and poor medium plus 2% casamino acids as indicated (richâpoor+aa). Growth was maintained in log phase conditions by regular dilutions in the corresponding media. Shown is the fraction of viable <i>ÎrsfA</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 (richârich) or poor M9 medium (richâpoor). 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 (S1âS6) and the relative amount of RsfA was quantified by Western-blot (represented with bars). S1âS3: samples were analyzed from both strains. S4âS6: 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 âŒ3 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>ÎrsfA</i> and WT strains; blue, the fraction (in %) of the mutant strain. (F) Expression of ÎČ-galactosidase as reporter to test translational activity of logarithmic and stationary phase cells in WT and <i>ÎrsfA</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
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