Regulation of RpoS by a novel small RNA: the characterization of RprA

Translational regulation of the stationary phase sigma factor RpoS is mediated by the formation of a double‐stranded RNA stem–loop structure in the upstream region of the rpoS messenger RNA, occluding the translation initiation site. The interaction of the rpoS mRNA with a small RNA, DsrA, disrupts the double‐strand pairing and allows high levels of translation initiation. We screened a multicopy library of Escherichia coli DNA fragments for novel activators of RpoS translation when DsrA is absent. Clones carrying rprA (RpoS regulator RNA) increased the translation of RpoS. The rprA gene encodes a 106 nucleotide regulatory RNA. As with DsrA, RprA is predicted to form three stem–loops and is highly conserved in Salmonella and Klebsiella species. Thus, at least two small RNAs, DsrA and RprA, participate in the positive regulation of RpoS translation. Unlike DsrA, RprA does not have an extensive region of complementarity to the RpoS leader, leaving its mechanism of action unclear. RprA is non‐essential. Mutations in the gene interfere with the induction of RpoS after osmotic shock when DsrA is absent, demonstrating a physiological role for RprA. The existence of two very different small RNA regulators of RpoS translation suggests that such additional regulatory RNAs are likely to exist, both for regulation of RpoS and for regulation of other important cellular components.

[1]  J. Foster,et al.  Effects of DksA and ClpP protease on sigma S production and virulence in Salmonella typhimurium , 1999, Molecular microbiology.

[2]  B. Sahagan,et al.  A small, unstable RNA molecule of Escherichia coli: spot 42 RNA: I. Nucleotide sequence analysis☆ , 1979 .

[3]  W. Benjamin,et al.  Avirulence of LT2 strains of Salmonella typhimurium results from a defective rpoS gene , 1997, Infection and immunity.

[4]  Regulation of Capsule Synthesis: Modification of the Two-Component Paradigm by an Accessory Unstable Regulator , 1995 .

[5]  J. A. Newitt,et al.  The E. coli Signal Recognition Particle Is Required for the Insertion of a Subset of Inner Membrane Proteins , 1997, Cell.

[6]  D. Apirion,et al.  The gene for a small stable RNA (10Sa RNA) of Escherichia coli , 1989, Molecular microbiology.

[7]  S. Gottesman,et al.  Clp P represents a unique family of serine proteases. , 1990, The Journal of biological chemistry.

[8]  R. Lease,et al.  A trans-acting RNA as a control switch in Escherichia coli: DsrA modulates function by forming alternative structures. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[9]  D. Jin,et al.  The rpoB mutants destabilizing initiation complexes at stringently controlled promoters behave like "stringent" RNA polymerases in Escherichia coli. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[10]  G. Storz,et al.  The OxyS regulatory RNA represses rpoS translation and binds the Hfq (HF‐I) protein , 1998, The EMBO journal.

[11]  G. Storz,et al.  A Small, Stable RNA Induced by Oxidative Stress: Role as a Pleiotropic Regulator and Antimutator , 1997, Cell.

[12]  M. Cusick,et al.  Riboregulation in Escherichia coli: DsrA RNA acts by RNA:RNA interactions at multiple loci. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[13]  N. Majdalani,et al.  DsrA RNA regulates translation of RpoS message by an anti-antisense mechanism, independent of its action as an antisilencer of transcription. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[14]  G. Storz,et al.  Small RNAs in Escherichia coli. , 1999, Trends in microbiology.

[15]  R. Hengge-aronis,et al.  Identification of transcriptional start sites and the role of ppGpp in the expression of rpoS, the structural gene for the sigma S subunit of RNA polymerase in Escherichia coli , 1995, Journal of bacteriology.

[16]  B. Sahagan,et al.  A small, unstable RNA molecule of Escherichia coli: spot 42 RNA. I. Nucleotide sequence analysis. , 1979, Journal of molecular biology.

[17]  S. Gottesman,et al.  A small RNA acts as an antisilencer of the H-NS-silenced rcsA gene of Escherichia coli. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[18]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[19]  H. Shuman,et al.  The Legionella pneumophila rpoS Gene Is Required for Growth within Acanthamoeba castellanii , 1999, Journal of bacteriology.

[20]  J. Belasco,et al.  1 – mRNA Degradation in Prokaryotic Cells: An Overview , 1993 .

[21]  T. Schweder,et al.  Regulation of Escherichia coli starvation sigma factor (sigma s) by ClpXP protease , 1996, Journal of bacteriology.

[22]  D. Belin,et al.  Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter , 1995, Journal of bacteriology.

[23]  R. Roy,et al.  Transcriptional induction of the conserved alternative sigma factor RpoS in Escherichia coli is dependent on BarA, a probable two‐component regulator , 2000, Molecular microbiology.

[24]  A. Zhang,et al.  Hfq Is Necessary for Regulation by the Untranslated RNA DsrA , 2001, Journal of bacteriology.

[25]  T. Elliott,et al.  Efficient translation of the RpoS sigma factor in Salmonella typhimurium requires host factor I, an RNA-binding protein encoded by the hfq gene , 1996, Journal of bacteriology.

[26]  Keith Dudley Short protocols in molecular biology , 1990 .

[27]  G. Storz,et al.  6S RNA Regulates E. coli RNA Polymerase Activity , 2000, Cell.

[28]  M. Pirhonen,et al.  Role of RpoS in virulence and stress tolerance of the plant pathogen Erwinia carotovora subsp. carotovora. , 1999, Microbiology.

[29]  Martin E. Mulligan,et al.  Analysis of the occurrence of promoter-sites in DNA , 1986, Nucleic Acids Res..

[30]  P. Neubauer,et al.  The General Stress Sigma Factor ςS ofEscherichia coli Is Induced during Diauxic Shift from Glucose to Lactose , 1998, Journal of bacteriology.

[31]  R. Miller,et al.  One-step preparation of competent Escherichia coli: transformation and storage of bacterial cells in the same solution. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[32]  R. Hengge-aronis,et al.  Interplay of global regulators and cell physiology in the general stress response of Escherichia coli. , 1999, Current opinion in microbiology.

[33]  R. Hengge-aronis,et al.  The RNA-binding protein HF-I, known as a host factor for phage Qbeta RNA replication, is essential for rpoS translation in Escherichia coli. , 1996, Genes & development.

[34]  R. Sauer,et al.  Role of a Peptide Tagging System in Degradation of Proteins Synthesized from Damaged Messenger RNA , 1996, Science.

[35]  M. Winkler,et al.  Characterization of broadly pleiotropic phenotypes caused by an hfq insertion mutation in Escherichia coli K‐12 , 1994, Molecular microbiology.

[36]  G. Church,et al.  Methods for generating precise deletions and insertions in the genome of wild-type Escherichia coli: application to open reading frame characterization , 1997, Journal of bacteriology.

[37]  S. Gottesman,et al.  Regulation of Proteolysis of the Stationary-Phase Sigma Factor RpoS , 1998, Journal of bacteriology.

[38]  R. Hengge-aronis,et al.  The cellular concentration of the sigma S subunit of RNA polymerase in Escherichia coli is controlled at the levels of transcription, translation, and protein stability. , 1994, Genes & development.

[39]  T. Schweder,et al.  Regulation ofEscherichia coliStarvation Sigma Factor (s s ) by ClpXP Protease , 1996 .

[40]  S. Gottesman,et al.  The small RNA, DsrA, is essential for the low temperature expression of RpoS during exponential growth in Escherichia coli. , 1996, The EMBO journal.

[41]  T. Elliott,et al.  Mutations that increase expression of the rpoS gene and decrease its dependence on hfq function in Salmonella typhimurium , 1997, Journal of bacteriology.

[42]  M. Bally,et al.  RpoS-dependent stress tolerance in Pseudomonas aeruginosa. , 1999, Microbiology.