Ms1 is a sRNA recently found in mycobacteria and several other actinobacterial species. Ms1 interacts with the RNA polymerase (RNAP) core devoid of sigma factors, which differs from 6S RNA that binds to RNAP holoenzymes containing the primary sigma factor. Here we show that Ms1 is the most abundant non-rRNA transcript in stationary phase in Mycobacterium smegmatis. The accumulation of Ms1 stems from its high-level synthesis combined with decreased degradation. We identify the Ms1 promoter, PMs1 , and cis-acting elements important for its activity. Furthermore, we demonstrate that PNPase (an RNase) contributes to the differential accumulation of Ms1 during growth. Then, by comparing the transcriptomes of wt and ΔMs1 strains from stationary phase, we reveal that Ms1 affects the intracellular level of RNAP. The absence of Ms1 results in decreased levels of the mRNAs encoding β and β' subunits of RNAP, which is also reflected at the protein level. Thus, the ΔMs1 strain has a smaller pool of RNAPs available when the transcriptional demand increases. This contributes to the inability of the ΔMs1 strain to rapidly react to environmental changes during outgrowth from stationary phase.

• MeSH
• bakteriální RNA metabolismus   MeSH
• delece genu MeSH
• DNA řízené RNA-polymerasy metabolismus   MeSH
• malá nekódující RNA genetika   metabolismus   MeSH
• Mycobacterium smegmatis enzymologie   genetika   růst a vývoj   metabolismus   MeSH
• stanovení celkové genové exprese MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Potato spindle tuber viroid (PSTVd) belongs to plant-pathogenic, circular, non-coding RNAs. Its propagation is accompanied by (mis)regulation of host genes and induction of pathogenesis symptoms including changes of leaf morphogenesis depending on the strength of viroid variant. We found strong genotype-dependent suppression of tomato morphogenesis-regulating transcription factor SANT/HTH-Myb (SlMyb) due to viroid pathogenesis. Its relative mRNA level was found to be significantly decreased in PSTVd-sensitive tomato (cvs Rutgers and Heinz 1706) due to degradation processes, but increased in PSTVd-tolerant (cv. Harzfeuer). In heterologous system of Nicotiana benthamiana, we observed a SlMyb-associated necrotic effect in agroinfiltrated leaf sectors during ectopic overexpression. Leaf sector necroses were accompanied by activation of nucleolytic enzymes but were suppressed by a strongly pathogenic PSTVd variant. Contrary to that, PSTVd's effect was inhibited by the silencing suppressor p19. It was found that in both, Solanum lycopersicum leaves and N. benthamiana leaf sectors, SlMyb mRNA degradation was significantly stronger in viroid-infected tissues. Necroses induction as well as gene silencing experiments using the SANT/HTH-Myb homologues revealed involvement of this Myb in physiological changes like distortions in flower morphogenesis and growth suppression.

• MeSH
• interakce hostitele a patogenu MeSH
• listy rostlin růst a vývoj   metabolismus   virologie   MeSH
• malá nekódující RNA genetika   metabolismus   MeSH
• messenger RNA genetika   metabolismus   MeSH
• molekulární sekvence - údaje MeSH
• nemoci rostlin genetika   virologie   MeSH
• regulátory růstu rostlin genetika   metabolismus   MeSH
• RNA virová genetika   metabolismus   MeSH
• rostlinné proteiny genetika   metabolismus   MeSH
• sekvenční analýza RNA MeSH
• Solanum lycopersicum genetika   metabolismus   virologie   MeSH
• tabák genetika   metabolismus   virologie   MeSH
• transkripční faktory genetika   metabolismus   MeSH
• viroidy genetika   patogenita   fyziologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Small RNAs (sRNAs) are molecules essential for a number of regulatory processes in the bacterial cell. Here we characterize Ms1, a sRNA that is highly expressed in Mycobacterium smegmatis during stationary phase of growth. By glycerol gradient ultracentrifugation, RNA binding assay, and RNA co-immunoprecipitation, we show that Ms1 interacts with the RNA polymerase (RNAP) core that is free of the primary sigma factor (σA) or any other σ factor. This contrasts with the situation in most other species where it is 6S RNA that interacts with RNAP and this interaction requires the presence of σA. The difference in the interaction of the two types of sRNAs (Ms1 or 6S RNA) with RNAP possibly reflects the difference in the composition of the transcriptional machinery between mycobacteria and other species. Unlike Escherichia coli, stationary phase M. smegmatis cells contain relatively few RNAP molecules in complex with σA. Thus, Ms1 represents a novel type of small RNAs interacting with RNAP.

• MeSH
• bakteriální chromozomy MeSH
• DNA řízené RNA-polymerasy metabolismus   MeSH
• konformace nukleové kyseliny MeSH
• malá nekódující RNA chemie   genetika   metabolismus   MeSH
• Mycobacterium smegmatis enzymologie   genetika   růst a vývoj   MeSH
• Mycobacterium genetika   MeSH
• sigma faktor metabolismus   MeSH
• syntenie MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

At low temperatures the Escherichia coli rpoS mRNA, encoding the stationary phase sigma factor RpoS, forms an intramolecular secondary structure (iss) that impedes translation initiation. Under these conditions the small RNA DsrA, which is stabilzed by Hfq, forms a duplex with rpoS mRNA sequences opposite of the ribosome-binding site (rbs). Both the DEAD box helicase CsdA and Hfq have been implicated in DsrA·rpoS duplex formation. Hfq binding to A-rich sequences in the rpoS leader has been suggested to restructure the mRNA, and thereby to accelerate DsrA·rpoS duplex formation, which, in turn, was deemed to free the rpoS rbs and to permit ribosome loading on the mRNA. Several experiments designed to elucidate the role of Hfq in DsrA-mediated translational activation of rpoS mRNA have been conducted in vitro. Here, we assessed RpoS synthesis in vivo to further study the role of Hfq in rpoS regulation. We show that RpoS synthesis was reduced when DsrA was ectopically overexpressed at 24 °C in the absence of Hfq despite of DsrA·rpoS duplex formation. This observation indicated that DsrA·rpoS annealing may not be sufficient for efficient ribosome loading on rpoS mRNA. In addition, a HfqG29A mutant protein was employed, which is deficient in binding to A-rich sequences present in the rpoS leader but proficient in DsrA binding. We show that DsrA·rpoS duplex formation occurs in the presence of the HfqG29A mutant protein at low temperature, whereas synthesis of RpoS was greatly diminished. RNase T1 footprinting studies of DsrA·rpoS duplexes in the absence and presence of Hfq or HfqG29A indicated that Hfq is required to resolve a stem-loop structure in the immediate coding region of rpoS mRNA. These in vivo studies corroborate the importance of the A-rich sequences in the rpoS leader and strongly suggest that Hfq, besides stabilizing DsrA and accelerating DsrA·rpoS duplex formation, is also required to convert the rpoS mRNA into a translationally competent form.

• MeSH
• 5' nepřekládaná oblast MeSH
• bakteriální proteiny genetika   metabolismus   MeSH
• bakteriální RNA genetika   MeSH
• Escherichia coli genetika   MeSH
• malá nekódující RNA genetika   metabolismus   MeSH
• messenger RNA metabolismus   MeSH
• mutace MeSH
• protein hostitelského faktoru 1 metabolismus   MeSH
• proteiny z Escherichia coli metabolismus   MeSH
• proteosyntéza MeSH
• regulace genové exprese u bakterií MeSH
• ribozomy metabolismus   MeSH
• sigma faktor genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH