Ribosome Shut-Down by 16S rRNA Fragmentation in Stationary-Phase Escherichia coli

Luidalepp, Hannes; Berger, Stefan; Joss, Oliver; Tenson, Tanel; Polacek, Norbert

doi:10.1016/j.jmb.2016.01.033

Cited by 18 publications

(16 citation statements)

References 41 publications

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“…Taken together, we found a substantial alteration in the rRNA/protein ratios of extracts from different in vivo backgrounds. Nevertheless, in contrary to other studies that detected a fragmentation of rRNA in stationary E. coli 41 , the CGE-LIF analysis showed only intact 16 S and 23 S rRNA (Supplemental Fig. S2 ), suggesting that rRNA integrity is conserved.…”

Section: Resultscontrasting

confidence: 99%

Cell-free protein synthesis from non-growing, stressed Escherichia coli

Failmezger

Rauter

Nitschel

et al. 2017

Sci Rep

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Cell-free protein synthesis is a versatile protein production system. Performance of the protein synthesis depends on highly active cytoplasmic extracts. Extracts from E. coli are believed to work best; they are routinely obtained from exponential growing cells, aiming to capture the most active translation system. Here, we report an active cell-free protein synthesis system derived from cells harvested at non-growth, stressed conditions. We found a downshift of ribosomes and proteins. However, a characterization revealed that the stoichiometry of ribosomes and key translation factors was conserved, pointing to a fully intact translation system. This was emphasized by synthesis rates, which were comparable to those of systems obtained from fast-growing cells. Our approach is less laborious than traditional extract preparation methods and multiplies the yield of extract per cultivation. This simplified growth protocol has the potential to attract new entrants to cell-free protein synthesis and to broaden the pool of applications. In this respect, a translation system originating from heat stressed, non-growing E. coli enabled an extension of endogenous transcription units. This was demonstrated by the sigma factor depending activation of parallel transcription. Our cell-free expression platform adds to the existing versatility of cell-free translation systems and presents a tool for cell-free biology.

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Section: Resultscontrasting

confidence: 99%

Cell-free protein synthesis from non-growing, stressed Escherichia coli

Failmezger

Rauter

Nitschel

et al. 2017

Sci Rep

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“…Both sites are exposed on the surface of the ribosome and the cleavage within helix 6 of 16S rRNA in the stationary phase ribosomes was recently reported. 49 As activation of MazF, 50,51 and the cleavage of 16S rRNA at #1500 ACA 28 were reported in stress conditions, we expected to identify the truncated 3 0 -end of 16S rRNA at A1499 and verify the presence of the stress ribosomes upon stress. Therefore, we applied 3 0 -RACE and Northern hybridization to analyze the RNA from stationary phase cells, CAM treated cultures and cultures where amino acid starvation was induced by mupirocin (MUP).…”

Section: Validation Of the Rna-seq Resultsmentioning

confidence: 94%

“…It was recently shown that cleavages within the helix 6 of 16S rRNA reduce the performance of stationary phase ribosomes. 49 At the same time, in many bacterial species, rRNA is normally discontinuous and is formed through excision of intervening sequences (IVS) which are located in the same variable helices. [59][60][61] Sequence tags have been inserted into these helices with no deleterious effects, 62 63 and when a Salmonella IVS was inserted into the helix 45 of E. coli 23S rRNA, it was excised by RNase III while the ribosomes retained functionality.…”

Section: Discussionmentioning

confidence: 99%

Toxins MazF and MqsR cleave Escherichia coli rRNA precursors at multiple sites

et al. 2016

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The endoribonuclease toxins of the E. coli toxin-antitoxin systems arrest bacterial growth and protein synthesis by targeting cellular mRNAs. As an exception, E. coli MazF was reported to cleave also 16S rRNA at a single site and separate an anti-Shine-Dalgarno sequence-containing RNA fragment from the ribosome. We noticed extensive rRNA fragmentation in response to induction of the toxins MazF and MqsR, which suggested that these toxins can cleave rRNA at multiple sites. We adapted differential RNA-sequencing to map the toxin-cleaved 5'- and 3'-ends. Our results show that the MazF and MqsR cleavage sites are located within structured rRNA regions and, therefore, are not accessible in assembled ribosomes. Most of the rRNA fragments are located in the aberrant ribosomal subunits that accumulate in response to toxin induction and contain unprocessed rRNA precursors. We did not detect MazF- or MqsR-cleaved rRNA in stationary phase bacteria and in assembled ribosomes. Thus, we conclude that MazF and MqsR cleave rRNA precursors before the ribosomes are assembled and potentially facilitate the decay of surplus rRNA transcripts during stress.

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“…This process, termed as ribosome hibernation, is thought to be a mechanism to fine-tune the translation process according to environmental conditions ( McKay and Portnoy, 2015 ). Recently, 16S rRNA fragmentation at the tip of helix 6 has been shown to attenuate the activity of 30S ribosomal subunit and thereby protein synthesis ( Luidalepp et al, 2016 ). Also, during limited nutrient availability, accumulation of truncated mRNA and deacylated tRNA occurs.…”

Section: Physiology Of the Stationary Phasementioning

confidence: 99%

Molecular Basis of Stationary Phase Survival and Applications

2017

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Stationary phase is the stage when growth ceases but cells remain metabolically active. Several physical and molecular changes take place during this stage that makes them interesting to explore. The characteristic proteins synthesized in the stationary phase are indispensable as they confer viability to the bacteria. Detailed knowledge of these proteins and the genes synthesizing them is required to understand the survival in such nutrient deprived conditions. The promoters, which drive the expression of these genes, are called stationary phase promoters. These promoters exhibit increased activity in the stationary phase and less or no activity in the exponential phase. The vectors constructed based on these promoters are ideal for large-scale protein production due to the absence of any external inducers. A number of recombinant protein production systems have been developed using these promoters. This review describes the stationary phase survival of bacteria, the promoters involved, their importance, regulation, and applications.

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Ribosome Shut-Down by 16S rRNA Fragmentation in Stationary-Phase Escherichia coli

Cited by 18 publications

References 41 publications

Cell-free protein synthesis from non-growing, stressed Escherichia coli

Cell-free protein synthesis from non-growing, stressed Escherichia coli

Toxins MazF and MqsR cleave Escherichia coli rRNA precursors at multiple sites

Molecular Basis of Stationary Phase Survival and Applications

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