title: Designing and using synthetic RNA thermometers for temperature-controlled gene expression in bacteria.
abstract: Many techniques have been developed for studying inducible gene expression, but all of them are multicomponent systems consisting of cis-acting elements at the DNA or RNA level, trans-acting regulator proteins and/or small molecules as inducers. RNA thermometers are the only known single-component regulators of gene expression. They consist of a temperature-sensitive secondary structure in the 5' untranslated region of the mRNA, which contains the ribosome-binding site. The ribosome-binding site can be masked or unmasked by a simple temperature shift, thereby repressing or inducing translation. Recently, we and others have designed synthetic RNA thermometers that are considerably simpler than naturally occurring thermometers and can be exploited as convenient on/off switches of gene expression. In this protocol, we describe the construction and use of synthetic RNA thermometers. We provide guidelines for the in silico design of thermometer-controlled mRNA leaders and for their experimental testing and optimization; the entire procedure can be completed in 2-3 weeks.
authors.affiliation: Max-Planck Institut für Molekulare Pflanzenphysiologie, Am Mühlenberg, Potsdam-Golm, Germany. rbock@mpimp-golm.mpg.de
authors.forename: Juliane
authors.initials: J
authors.lastname: Neupert
authors.forename: Ralph
authors.initials: R
authors.lastname: Bock
mesh_terms: D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005818:Genetic Engineering / Q000379:methods*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000138:chemical synthesis; D012313:RNA / Q000138:chemical synthesis* / Q000737:chemistry / Q000235:genetics*; D013696:Temperature; D013821:Thermometers
chemical_list: D009841:Oligonucleotides; D012313:RNA
references.citation: Plant J. 2009 Feb;57(3):436-45
references.pmid: 18939966
references.citation: Nucleic Acids Res. 1994 Nov 25;22(23):4953-7
references.pmid: 7528374
references.citation: Gene. 2005 Nov 21;361:13-37
references.pmid: 16213112
references.citation: Gene. 1999 Jul 8;234(2):187-208
references.pmid: 10395892
references.citation: Science. 1994 Feb 11;263(5148):802-5
references.pmid: 8303295
references.citation: Nucleic Acids Res. 2008 Nov;36(19):e124
references.pmid: 18753148
references.citation: Genes Dev. 2008 Dec 15;22(24):3383-90
references.pmid: 19141470
references.citation: Nature. 2006 Jun 29;441(7097):1167-71
references.pmid: 16728979
references.citation: J Bacteriol. 1986 Jul;167(1):415-9
references.pmid: 2941413
references.citation: Anal Biochem. 1987 Nov 1;166(2):368-79
references.pmid: 2449095
references.citation: Trends Genet. 1994 Nov;10(11):402-7
references.pmid: 7809946
references.citation: Science. 2004 Oct 8;306(5694):275-9
references.pmid: 15472076
references.citation: Genes Dev. 1999 Mar 15;13(6):655-65
references.pmid: 10090722
references.citation: Proc Natl Acad Sci U S A. 2003 Nov 11;100(23):13213-8
references.pmid: 14585931
references.citation: FEMS Microbiol Rev. 2006 Jan;30(1):3-16
references.pmid: 16438677
references.citation: J Biol Chem. 2003 Nov 28;278(48):47915-21
references.pmid: 12963744
references.citation: Plant Biotechnol J. 2008 Dec;6(9):897-913
references.pmid: 19548344
references.citation: Nature. 1970 Aug 15;227(5259):680-5
references.pmid: 5432063
references.citation: Nat Rev Genet. 2007 Oct;8(10):776-90
references.pmid: 17846637
references.citation: Nat Rev Mol Cell Biol. 2004 Jun;5(6):451-63
references.pmid: 15173824
references.citation: Genes Dev. 1999 Mar 15;13(6):633-6
references.pmid: 10090718
references.citation: Mol Genet Genomics. 2007 Nov;278(5):555-64
references.pmid: 17647020
references.citation: Proc Natl Acad Sci U S A. 2004 Apr 27;101(17):6421-6
references.pmid: 15096624
references.citation: Nucleic Acids Res. 2001 Dec 1;29(23):4800-7
references.pmid: 11726689
references.citation: Nucleic Acids Res. 2003 Jul 1;31(13):3406-15
references.pmid: 12824337
references.citation: J Mol Biol. 1983 Jun 5;166(4):557-80
references.pmid: 6345791
references.citation: Biol Chem. 2008 Oct;389(10):1319-26
references.pmid: 18713019
references.citation: Cell. 2002 Sep 6;110(5):551-61
references.pmid: 12230973
references.citation: Annu Rev Microbiol. 2005;59:487-517
references.pmid: 16153177
references.citation: Nature. 2002 Oct 31;419(6910):952-6
references.pmid: 12410317
references.citation: EMBO J. 2006 Jun 7;25(11):2487-97
references.pmid: 16710302
references.citation: Nucleic Acids Res. 1979 Nov 24;7(6):1513-23
references.pmid: 388356