The cellular synthesis machinery runs at high speed in the exponential (logarithmic) phase of bacterial growth. The growth rate slows in semi-log phase and finally comes to a halt at higher cell density in the stationary phase, usually caused by nutrient depletion. Several bacterial factors bind to ribosomes and thus support the dormant state of the ribosomes in the stationary phase, such as the ribosome modulation factor (RMF), hibernation promoting factor (HPF) or stationary-phase-induced ribosome-associated protein (SRA) 
. RMF (homologues exist only in the γ-proteobacteria) alone or together with the more broadly distributed HPF are essential for the formation of 70S dimers in the stationary phase, so called 100S particles; an inactivation of the RMF gene causes a viability defect at prolonged periods in stationary phase 
. Phenotypical effects of knock-out strains concerning the other factors have not been reported.
A first analysis of RsfA-binding partners identified a group of proteins including a number of ribosomal proteins 
. Similarly, other groups suggested various ribosomal proteins as binding partners 
, the common denominator being that all proteins were derived from the large subunit. Thorough analyses presented here identified the ribosomal protein L14 as the docking station (, ), and mutation of conserved amino acid residues of L14 at the surface of this protein abolished RsfA binding, clearly demonstrating L14 as the binding protein (). Interestingly, the three most conserved residues of RsfA as shown by the multiple sequence alignment (Figure S1A
) are located at the interface with L14 predicted by docking. The three residues are W120, D124 and R140 (alignment numbers), corresponding to residue numbers W77, D81 and R95 in E. coli
RsfA. D81 is predicted to be in direct contact with R98 of L14 that was shown to disrupt the interaction when mutated. Another such critical residue, K114 of L14, is predicted to be in contact with a fairly conserved residue with RsfA L103 (position 148 in the alignment).
The only other known protein that like RsfA also docks to the ribosomal protein L14 of eukaryotic ribosomes is the so-called initiation factor eIF6, which is not a homologue to RsfA and is thought to block ribosome association in archaea and in eukaryotes from yeast to man 
. However, in eukaryotes eIF6 is rather a 60S assembly factor and plays an essential role in the late pre-25S rRNA processing and the export of the 60S subunit from the nucleolus to the cytoplasm 
. Depletion of eIF6 is eventually lethal, in contrast to RsfA. Interestingly, eIF6 is restricted to the eukaryotic nucleus/cytoplasm and to archaea 
, while RsfA is present in almost all bacteria and their descendent eukaryotic organelles ().
Studies with the human mitochondrial homologue of RsfA, C7orf30, have recently suggested that this protein is involved in ribosomal assembly and/or translation 
. Our results do not indicate any assembly defects as deletion strains of rsfA
appear to have perfectly assembled ribosomes (sucrose gradients not shown) and actually translate as well as wild type strains at logarithmic phase (). In addition, we could show that C7orf30 inhibits translation by mitochondrial ribosomes (). It remains possible that C7orf30 has multiple roles in mitochondria or that its role in ribosome assembly is indirect.
In rich medium bacterial cells produce proteins at maximum rates to sustain cell division. Furthermore, bacterial cells take up many metabolic precursors such as amino acids and thus block corresponding synthesis pathways. In contrast, in poor/minimal medium protein synthesis must be down-regulated in a concerted fashion in order to save energy and resources, and at the same time many synthesis pathways such as those for the synthesis of amino acids have to be switched on 
. The results presented here suggest that RsfA plays a prominent role in this down-regulation by silencing ribosome activities. We observe two strong phenotypes with the ΔrsfA
strain: (i) the viability is strongly impaired in the stationary phase () and (ii) after a transition from rich to poor media the adaptation phase lasts more than 10 hours before resuming growth again in striking contrast to WT cells (), which overgrow the mutant strain in a few generations. Just adding casamino acids to the minimal medium relieves the strong growth defects of the ΔrsfA
strain (). Adding amino acids will switch off most of the amino-acid synthesis pathways similar to the situation during the logarithmic phase in the presence of rich medium, when the silencing effect of RsfA is not strictly required. In contrast, during starvation and in the absence of ribosomal silencing (ΔrsfA
), energy would be wasted affecting the conversion of the metabolic network, eventually causing deleterious growth defects. Accordingly, protein synthesis is seriously attenuated in the stationary phase, when RsfA is present (i.e. wild type cells) in contrast to protein synthesis in the ΔrsfA
strain (). Attenuation of protein synthesis by RsfA seems to be of utmost importance for reorganization the metabolic state on the way to the stationary phase, since the absence of this factor threatens seriously the viability in the stationary phase (), and it explains the well-known effect that ribosomes are much less active, when derived from the stationary rather than from log-phase cells 
When RsfA is added to ribosomal subunits it blocks 70S formation and thus protein synthesis (), whereas the factor does not interfere with the elongation phase of protein synthesis when added to ribosomes that have passed the initiation phase (, right panel). We conclude that RsfA, as a ribosomal silencing factor, is damping the translational activity under restricted energy (stationary phase) or nutrient conditions (growth in poor medium) thus harmonizing translation with the general metabolic state, i.e.
RsfA works in line with the stringent response 
and thus plays a key role in the physiology of the stationary phase and the translational adaptation during the transition from rich to poor medium.
Our experiments suggest a direct silencing effect of RsfA sketched in : when the ribosomal activity should be silenced, RsfA binds to the ribosomal protein L14 at the interface of the large subunit and by impairing association of the ribosomal subunits translation is hampered. We demonstrated that RsfA damps the ribosomal elongation in bacterial and mammalian mitochondrial systems (). The importance of RsfA in eukaryotic organelles is indicated by the fact that a mutation in the gene of the RsfA orthologue Iojap in Zea mays
leads to irregular albino patterns on maize leafs and germless seeds due to failure of proplastids to differentiate into chloroplasts 
. Photosynthesis and respiration can vary enormously in plastids and mitochondria, respectively, and as suggested by the experiment shown in , the RsfA orthologue might accordingly regulate protein synthesis in these organelles using the mechanism suggested here.