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5S rRNA is an indispensable component of cytoplasmic ribosomes in all species. The functions of 5S rRNA and the reasons for its evolutionary preservation as an independent molecule remain unclear. Here we used ribosome engineering to investigate whether 5S rRNA autonomy is critical for ribosome function and cell survival. By linking circularly permutated 5S rRNA with 23S rRNA we generated a bacterial strain devoid of free 5S rRNA. Viability of the engineered cells demonstrates that autonomous 5S rRNA is dispensable for cell growth under standard conditions and is unlikely to have essential functions outside the ribosome. The fully assembled ribosomes carrying 23S-5S rRNA are highly active in translation. However, the engineered cells accumulate aberrant 50S subunits unable to form stable 70S ribosomes. Cryo-EM analysis revealed a malformed peptidyl transferase center in the misassembled 50S subunits. Our results argue that the autonomy of 5S rRNA is preserved due to its role in ribosome biogenesis.

Ribosome degradation has been extensively described from the viewpoint of its main component, rRNA. Here, we aim to complement our knowledge by quantitatively analyzing r-protein degradation and stability both in the ribosomes and in the whole-cell proteome during stationary phase in E. coli . r-proteins are considered to be very stable in the proteome.

Ribosomes are essential macromolecular complexes conducting protein biosynthesis in all domains of life. Cells can have heterogeneous ribosomes, i.e. ribosomes with various ribosomal RNA and ribosomal protein (r-protein) composition. However, the functional importance of heterogeneous ribosomes has remained elusive. One of the possible sources for ribosome heterogeneity is provided by paralogous r-proteins. In E. coli, ribosomal protein bL31 has two paralogs: bL31A encoded by rpmE and bL31B encoded by ykgM. This study investigates phenotypic effects of these ribosomal protein paralogs using bacterial strains expressing only bL31A or bL31B. We show that bL31A confers higher fitness to E. coli under lower temperatures. In addition, bL31A and bL31B have different effects on translation reading frame maintenance and apparent translation processivity in vivo as demonstrated by dual luciferase assay. In general, this study demonstrates that ribosomal protein paralog composition (bL31A versus bL31B) can affect cell growth and translation outcome. To survive, grow and reproduce organisms need proteins that function as enzymes, messengers, structural components of intra-and intercellular structures, transport and storage molecules. In addition to proper functionality, proteins have to be available in cells at the right time, place and in adequate amount to ensure/contribute to organism's fitness/viability. Therefore, regulation of protein synthesis-translation-is of central importance in molecular biology. Translation is a highly complex process comprising an mRNA, aminoacylated tRNA-s, several protein factors and ribosomes and it can be regulated by various mechanisms 1,2. An emerging paradigm is ribosome composition mediated translational control 3 that is based on the hypothesis that ribosome can regulate translation via its composition. This concept is based on the accumulating evidence that eukaryotic and bacterial cells produce heterogeneous ribosomes, i.e. ribosomes containing alternative components 4,5. Ribosome heterogeneity has various sources: rRNA sequence and modifications, ribosomal protein (r-protein) stoichiometry, modifications and paralogs 6. Therefore, the potential of creating ribosomes with various composition is enormous 7. An intriguing question is whether ribosome heterogeneity is functional. It means that first, biochemically different ribosomes are present in vivo under adverse growth conditions and second, that these variant ribosomes affect cell physiology via protein synthesis 8. Recently, functional ribosome heterogeneity has been defined as "variations in ribosome composition that influence its activity, thereby changing the output of translation" 9. As translation initiation is the main regulatory checkpoint for protein synthesis most research on the functionality of ribosome heterogeneity has been focused on this step of translation 6. Specifically, one possible viewpoint to ribosome composition mediated translational control has been ribosome filter hypothesis stating that ribosomes may d...

Bacterial ribosomes contain over 50 ribosome core proteins (r-proteins). Tens of non-ribosomal proteins bind to ribosomes to promote various steps of translation or suppress protein synthesis during ribosome hibernation. This study sets out to determine how translation activity is regulated during the prolonged stationary phase. Here, we report the protein composition of ribosomes during the stationary phase. According to quantitative mass-spectrometry analysis, ribosome core proteins bL31B and bL36B are present during the late log and first days of the stationary phase and are replaced by corresponding A paralogs later in the prolonged stationary phase. Ribosome hibernation factors Rmf, Hpf, RaiA, and Sra are bound to the ribosomes during the onset and a few first days of the stationary phase when translation is strongly suppressed. In the prolonged stationary phase, a decrease in ribosome concentration is accompanied by an increase in translation and association of translation factors with simultaneous dissociation of ribosome hibernating factors. The dynamics of ribosome-associated proteins partially explain the changes in translation activity during the stationary phase.

Bacterial ribosomes are composed of three RNA (rRNA) and over 50 ribosomal protein (r-protein) molecules. R-proteins are essential for ribosome assembly, structural stability, and also participate in almost all ribosome functions. Ribosomal components are present in stoichiometric amounts in the mature 70S ribosomes during the exponential and early stationary growth phases. Ribosomes are degraded in stationary phase, however, the stability and fate of ribosomal proteins during stationary growth phase are not known. In this study, we report a quantitative analysis of ribosomal components during the extended stationary phase in Escherichia coli. We show that: (a) the quantity of ribosomes per cell decreases in the stationary phase. (b) 70S ribosomes contain r-proteins in stoichiometric amounts. (c) 30S subunits are degraded faster than 50S. (d) In total proteome, the quantity of 20 r-proteins decreases during 14 days (short-lived r-proteins) concomitantly with the reduction of cellular RNA. (e) 30 r-proteins are stable and form a pool of free r-proteins (stable r-proteins). Thus r-proteins are present in non-stochiometric amounts in the proteome of E. coli during the extended stationary phase.

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