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

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.

The effects of disruptions in ribosomal active sites and in intersubunit contacts on ribosomal degradation in Escherichia coli.

Although ribosomes are very stable under most conditions, ribosomal degradation does occur in diverse groups of organisms in response to specific stresses or environmental conditions. While non-functional ribosome decay (NRD) in yeast is well characterized, very little is known of the mechanisms that initiate ribosomal degradation in bacteria. Here we test ribosome degradation in growing Escherichia coli expressing mutant ribosomes. We found that mutations in the 16S rRNA decoding centre (G530U and A1492C) and 23S rRNA active site (A2451G) do not lead to ribosomal degradation. In contrast, 23S rRNA mutation U2585A causes degradation of both the large and small ribosomal subunits in E. coli. We further tested mutations in 23S rRNA, which disrupt ribosomal intersubunit bridges B2a and B3. Deletion of helix 69 of 23S rRNA and the point mutation A1912G in the same helix did not destabilize ribosomes, while expression of mutations A1919G in H69 and A1960G in H71 led to degradation of both mutant and wild-type ribosomes. Our results suggest an actively induced mechanism requiring de novo protein synthesis for ribosomal degradation in E. coli, which degrades both structurally inactive and active ribosomes.

When stable RNA becomes unstable: the degradation of ribosomes in bacteria and beyond.

This review takes a comparative look at the various scenarios where ribosomes are degraded in bacteria and eukaryotes with emphasis on studies involving Escherichia coli and Saccharomyces cerevisiae. While the molecular mechanisms of degradation in bacteria and yeast appear somewhat different, we argue that the underlying causes of ribosome degradation are remarkably similar. In both model organisms during ribosomal assembly, partially formed pre-ribosomal particles can be degraded by at least two different sequentially-acting quality control pathways and fully assembled but functionally faulty ribosomes can be degraded in a separate quality control pathway. In addition, ribosomes that are both structurally- and functionally-sound can be degraded as an adaptive measure to stress.

Ribosome degradation in growing bacteria.

Ribosomes are large ribozymes that synthesize all cellular proteins. As protein synthesis is rate-limiting for bacterial growth and ribosomes can comprise a large portion of the cellular mass, elucidation of ribosomal turnover is important to the understanding of cellular physiology. Although ribosomes are widely believed to be stable in growing cells, this has never been rigorously tested, owing to the lack of a suitable experimental system in commonly used bacterial model organisms. Here, we develop an experimental system to directly measure ribosomal stability in Escherichia coli. We show that (i) ribosomes are stable when cells are grown at a constant rate in the exponential phase; (ii) more than half of the ribosomes made during exponential growth are degraded during slowing of culture growth preceding the entry into stationary phase; and (iii) ribosomes are stable for many hours in the stationary phase. Ribosome degradation occurs in growing cultures that contain almost no dead cells and coincides with a reduction of comparable magnitude in the cellular RNA concentration.

Expression of heat shock proteins and nitrotyrosine in small arteries from patients with coronary heart disease.

Heat shock proteins (HSPs) have been suggested to play an important role in the pathogenesis of cardiovascular disease; however, their levels in resistance arteries and their role as useful markers for endothelial dysfunction are not well known. In this paper we studied the levels of HSP90, HSP70, HSP60, HSP27, and of the oxidative stress marker nitrotyrosine (NT) in isolated small subcutaneous arteries from female and male patients with coronary heart disease (CHD) and compared them with healthy controls. HSPs and NT levels were analyzed by immunohistochemistry (IHC) with streptavidin-biotin complex and 3,3'-diaminobenzidine (DAB) staining. The results were assessed with a semi-quantitative method. The study showed lower levels of HSP90 in arteries from both male and female patients when compared to the healthy controls, while levels of HSP70 were lower only in male patients versus controls. The levels of HSP60 and HSP27 did not show any significant difference in either the male or the female groups. NT levels were higher in the arteries from female patients as compared to controls. In conclusion, the present study strengthens the concept that HSPs may play an important role in the pathogenesis of CHD, and that at least two of them, HSP70 and HSP90, may have useful applications as markers of vascular dysfunction in resistance arteries.