Molecular Genetics and Genomics

, Volume 278, Issue 5, pp 555–564 | Cite as

Genome-wide bioinformatic prediction and experimental evaluation of potential RNA thermometers

  • Torsten Waldminghaus
  • Lena C. Gaubig
  • Franz NarberhausEmail author
Original Paper


Only recently, the fundamental role of regulatory RNAs in prokaryotes and eukaryotes has been appreciated. We developed a pipeline from bioinformatic prediction to experimental validation of new RNA thermometers. Known RNA thermometers are located in the 5′-untranslated region of certain heat shock or virulence genes and control translation by temperature-dependent base pairing of the ribosome binding site. We established the searchable database RNA-SURIBA (Structures of Untranslated Regions In BActeria). A structure-based search pattern reliably recognizes known RNA thermometers and predicts related structures upstream of annotated genes in complete genome sequences. The known ROSE1 (Repression Of heat Shock gene Expression) thermometer and several other functional ROSE-like elements were correctly predicted. For further investigation, we chose a new candidate upstream of the phage shock gene D (pspD) in the pspABCDE operon of E. coli. We established a new reporter gene system that measures translational control at heat shock temperatures and we demonstrated that the upstream region of pspD does not confer temperature control to the phage shock gene. However, translational efficiency was modulated by a point mutation stabilizing the predicted hairpin. Testing other candidates by this structure prediction and validation process will lead to new insights into the requirements for biologically active RNA thermometers. The database is available on


Posttranscriptional regulation Reporter system Riboswitch RNA structure RNA thermometer 



We are grateful to Jan Tommassen (Utrecht) for antisera against Psp proteins and for plasmids pJP380 and pJF119HE, and Wolfgang Schumann (Bayreuth) for vector pGF-bgaB. We thank Michael Zuker (Rensselaer Polytechnic Institute, Troy, USA) for providing us with a licence of the mfold program and Juliane Alfsmann (Bochum) for construction of some bgaB fusions. This work was funded by German Research Foundation (DFG NA240/4).

Supplementary material

438_2007_272_MOESM1_ESM.doc (64 kb)
Table S1: Genomes included in the database RNA-SURIBA (DOC 64 kb)
438_2007_272_MOESM2_ESM.doc (62 kb)
Table S2: Parameters contained in the database RNA-SURIBA (DOC 63 kb)


  1. Adams H, Teertstra W, Demmers J, Boesten R, Tommassen J (2003) Interactions between phage-shock proteins in Escherichia coli. J Bacteriol 185:1174–1180PubMedCrossRefGoogle Scholar
  2. Altuvia S, Kornitzer D, Teff D, Oppenheim AB (1989) Alternative mRNA structures of the cIII gene of bacteriophage lambda determine the rate of its translation initiation. J Mol Biol 210:265–280PubMedCrossRefGoogle Scholar
  3. Barrick JE et al. (2004) New RNA motifs suggest an expanded scope for riboswitches in bacterial genetic control. Proc Natl Acad Sci USA 101:6421–6426PubMedCrossRefGoogle Scholar
  4. Brissette JL, Weiner L, Ripmaster TL, Model P (1991) Characterization and sequence of the Escherichia coli stress-induced psp operon. J Mol Biol 220:35–48PubMedCrossRefGoogle Scholar
  5. Chowdhury S, Ragaz C, Kreuger E, Narberhaus F (2003) Temperature-controlled structural alterations of an RNA thermometer. J Biol Chem 278:47915–47921PubMedCrossRefGoogle Scholar
  6. Chowdhury S, Maris C, Allain FH, Narberhaus F (2006) Molecular basis for temperature sensing by an RNA thermometer. EMBO J 25:2487–2497PubMedCrossRefGoogle Scholar
  7. Corbino KA et al (2005) Evidence for a second class of S-adenosylmethionine riboswitches and other regulatory RNA motifs in alpha-proteobacteria. Genome Biol 6:R70PubMedCrossRefGoogle Scholar
  8. Darwin AJ (2005) The phage-shock-protein response. Mol Microbiol 57:621–628PubMedCrossRefGoogle Scholar
  9. de Smit MH, van Duin J (1990) Secondary structure of the ribosome binding site determines translational efficiency: a quantitative analysis. Proc Natl Acad Sci USA 87:7668–7672PubMedCrossRefGoogle Scholar
  10. de Smit MH, van Duin J (1994) Control of translation by mRNA secondary structure in Escherichia coli. A quantitative analysis of literature data. J Mol Biol 244:144–150PubMedCrossRefGoogle Scholar
  11. Eddy SR (2002) A memory-efficient dynamic programming algorithm for optimal alignment of a sequence to an RNA secondary structure. BMC Bioinformatics 3:18PubMedCrossRefGoogle Scholar
  12. Fogel GB et al. (2002) Discovery of RNA structural elements using evolutionary computation. Nucleic Acids Res 30:5310–5317PubMedCrossRefGoogle Scholar
  13. Guzman LM, Belin D, Carson MJ, Beckwith J (1995) Tight regulation, modulation, and high-level expression by vectors containing the arabinose pBAD promoter. J Bacteriol 177:4121–4130PubMedGoogle Scholar
  14. Hirata H, Negoro S, Okada H (1984) Molecular basis of isozyme formation of beta-galactosidases in Bacillus stearothermophilus: isolation of two beta-galactosidase genes, bgaA and bgaB. J Bacteriol 160:9–14PubMedGoogle Scholar
  15. Imai Y, Matsushima Y, Sugimura T, Terada M (1991) A simple and rapid method for generating a deletion by PCR. Nucleic Acids Res 19:2785PubMedCrossRefGoogle Scholar
  16. Johansson J, Mandin P, Renzoni A, Chiaruttini C, Springer M, Cossart P (2002) An RNA thermosensor controls expression of virulence genes in Listeria monocytogenes. Cell 110:551–561PubMedCrossRefGoogle Scholar
  17. Klein RJ, Eddy SR (2003) RSEARCH: finding homologs of single structured RNA sequences. BMC Bioinform 4:44CrossRefGoogle Scholar
  18. Mandal M, Breaker RR (2004) Gene regulation by riboswitches. Nat Rev Mol Cell Biol 5:451–463PubMedCrossRefGoogle Scholar
  19. Miller JH (1972) Experiments in molecular genetics. Cold Spring Harbor, New YorkGoogle Scholar
  20. Model P, Jovanovic G, Dworkin J (1997) The Escherichia coli phage-shock-protein (psp) operon. Mol Microbiol 24:255–261PubMedCrossRefGoogle Scholar
  21. Morita MT, Tanaka Y, Kodama TS, Kyogoku Y, Yanagi H, Yura T (1999) Translational induction of heat shock transcription factor σ32: evidence for a built-in RNA thermosensor. Genes Dev 13:655–665PubMedGoogle Scholar
  22. Nahvi A, Barrick JE, Breaker RR (2004) Coenzyme B12 riboswitches are widespread genetic control elements in prokaryotes. Nucleic Acids Res 32:143–150PubMedCrossRefGoogle Scholar
  23. Nakahigashi K, Yanagi H, Yura T (1995) Isolation and sequence analysis of rpoH genes encoding σ32 homologs from gram negative bacteria: conserved mRNA and protein segments for heat shock regulation. Nucleic Acids Res 23:4383–4390PubMedGoogle Scholar
  24. Narberhaus F, Käser R, Nocker A, Hennecke H (1998) A novel DNA element that controls bacterial heat shock gene expression. Mol Microbiol 28:315–323PubMedCrossRefGoogle Scholar
  25. Narberhaus F, Waldminghaus T, Chowdhury S (2006) RNA thermometers. FEMS Microbiol Rev 30:3–16PubMedCrossRefGoogle Scholar
  26. Nocker A, Hausherr T, Balsiger S, Krstulovic NP, Hennecke H, Narberhaus F (2001) A mRNA-based thermosensor controls expression of rhizobial heat shock genes. Nucleic Acids Res 29:4800–4807PubMedCrossRefGoogle Scholar
  27. Norrander J, Kempe T, Messing J (1983) Construction of improved M13 vectors using oligodeoxynucleotide-directed mutagenesis. Gene 26:101–106PubMedCrossRefGoogle Scholar
  28. Nudler E, Mironov AS (2004) The riboswitch control of bacterial metabolism. Trends Biochem Sci 29:11–17PubMedCrossRefGoogle Scholar
  29. Pridmore RD (1987) New and versatile cloning vectors with kanamycin-resistance marker. Gene 56:309–312PubMedCrossRefGoogle Scholar
  30. Rodionov DA, Vitreschak AG, Mironov AA, Gelfand MS (2002) Comparative genomics of thiamin biosynthesis in procaryotes. New genes and regulatory mechanisms. J Biol Chem 277:48949–48959PubMedCrossRefGoogle Scholar
  31. Sambrook JE, Fritsch F, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor, New YorkGoogle Scholar
  32. Schägger H, von Jagow G (1987) Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem 166:368–379PubMedCrossRefGoogle Scholar
  33. Schyns G, Potot S, Geng Y, Barbosa TM, Henriques A, Perkins JB (2005) Isolation and characterization of new thiamine-deregulated mutants of Bacillus subtilis. J Bacteriol 187:8127–8136PubMedCrossRefGoogle Scholar
  34. Stoss O, Mogk A, Schumann W (1997) Integrative vector for constructing single-copy translational fusions between regulatory regions of Bacillus subtilis and the bgaB reporter gene encoding a heat-stable beta-galactosidase. FEMS Microbiol Lett 150:49–54PubMedCrossRefGoogle Scholar
  35. Straney R, Krah R, Menzel R (1994) Mutations in the -10 TATAAT sequence of the gyrA promoter affect both promoter strength and sensitivity to DNA supercoiling. J Bacteriol 176:5999–6006PubMedGoogle Scholar
  36. Waldminghaus T, Fippinger A, Alfsmann J, Narberhaus F (2005) RNA thermometers are common in α- and γ-proteobacteria. Biol Chem 386:1279–1286PubMedCrossRefGoogle Scholar
  37. Waldminghaus T, Heidrich N, Brantl S, Narberhaus F (2007) FourU—a novel type of RNA thermometer in Salmonella. Mol Microbiol 65:413–424PubMedCrossRefGoogle Scholar
  38. Weiner L, Brissette JL, Model P (1991) Stress-induced expression of the Escherichia coli phage shock protein operon is dependent on σ54 and modulated by positive and negative feedback mechanisms. Genes Dev 5:1912–1923PubMedCrossRefGoogle Scholar
  39. Winkler WC, Breaker RR (2005) Regulation of bacterial gene expression by riboswitches. Annu Rev Microbiol 59:487–517PubMedCrossRefGoogle Scholar
  40. Zuker M (2003) Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res 31:3406–3415PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2007

Authors and Affiliations

  • Torsten Waldminghaus
    • 1
  • Lena C. Gaubig
    • 1
  • Franz Narberhaus
    • 1
    Email author
  1. 1.Lehrstuhl für Biologie der MikroorganismenRuhr-Universität BochumBochumGermany

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