SUMMARY Many bacteria contain an ortholog of the Ro autoantigen, a ring-shaped protein that binds noncoding RNAs (ncRNAs) called Y RNAs. In the only studied bacterium, Deinococcus radiodurans, the Ro ortholog Rsr functions in heat stress-induced rRNA maturation and starvation-induced rRNA decay. However, the mechanism by which this conserved protein and its associated ncRNAs act has been obscure. We report that Rsr and the exoribonuclease polynucleotide phosphorylase (PNPase) form an RNA degradation machine that is scaffolded by Y RNA. Single-particle electron microscopy, followed by docking of atomic models into the reconstruction, suggests that Rsr channels single-stranded RNA into the PNPase cavity. Biochemical assays reveal that Rsr and Y RNA adapt PNPase for effective degradation of structured RNAs. A Ro ortholog and ncRNA also associate with PNPase in Salmonella Typhimurium. Our studies identify a new ribonucleoprotein machine and demonstrate that ncRNA, by tethering a protein cofactor, can alter the substrate specificity of an enzyme.

UV light induces the expression of a wide variety of genes. At present, it is unclear how cells sense the extent of DNA damage and alter the expression of UV-induced genes appropriately. UV light induces DNA damage that blocks transcription, and the probability that a gene sustains transcription-blocking DNA damage is proportional to locus size and dose of UV light. Using colon carcinoma cells that express a temperature-sensitive variant of p53 and undergo p53-dependent apoptosis after UV irradiation, we found that the number of p53-induced genes identified by oligonucleotide microarray analysis decreased in a UV dose-dependent manner. This was associated with a statistically significant shift in the spectrum of p53-induced genes toward compact genes with fewer and smaller introns. Genes encoding proapoptotic proteins involved in the initiation of the mitochondrial apoptotic cascade were prominent among the compact p53 target genes, whereas genes encoding negative regulators of p53 and the mitochondrial apoptotic pathway were significantly larger. We propose that the shift in spectrum of UV-responsive gene expression caused by passive effects of UV lesions on transcription acts as a molecular dosimeter, ensuring the elimination of cells sustaining irreparable transcription-blocking DNA damage.DNA damage ͉ microarray ͉ p53 ͉ transcription ͉ stress responses

Small molecules play crucial roles in every major cellular process. Despite this, detecting their levels within cells remains a significant challenge. Here, we describe intracellular sensors of coenzyme B(12) that make use of the exquisite molecular detection capabilities of a naturally occurring riboswitch. These probes sensitively detect their target using colorimetric, fluorescent, or luminescent reporters. To assess their utility in the study of biological systems, the sensors were applied to examine the synthesis and the import of coenzyme B(12). The sensors were able to monitor the effects of genetic deletions, recombinant expression of foreign genes, and varied growth conditions on both of these processes. These results indicate that riboswitch-based sensors can provide valuable information on intracellular small molecule concentrations that can be employed in the study of related cellular processes.

Bacterial toxins with an AB 5 architecture consist of an active (A) subunit inserted into a ring-like platform comprised of five delivery (B) subunits. Salmonella Typhi, the cause of typhoid fever, produces an unusual A 2 B 5 toxin known as typhoid toxin. Here, we report that upon infection of human cells, S . Typhi produces two forms of typhoid toxin that have distinct delivery components but share common active subunits. The two typhoid toxins exhibit different trafficking properties, elicit different effects when administered to laboratory animals, and are expressed using different regulatory mechanisms and in response to distinct metabolic cues. Collectively, these results indicate that the evolution of two typhoid toxin variants has conferred functional versatility to this virulence factor. More broadly, this study reveals a new paradigm in toxin biology and suggests that the evolutionary expansion of AB 5 toxins was likely fueled by the plasticity inherent to their structural design coupled to the functional versatility afforded by the combination of homologous toxin components.