title: Ligand-induced folding of the thiM TPP riboswitch investigated by a structure-based fluorescence spectroscopic approach.
abstract: Riboswitches are genetic control elements within non-coding regions of mRNA. They consist of a metabolite-sensitive aptamer and an adjoining expression platform. Here, we describe ligand-induced folding of a thiamine pyrophosphate (TPP) responsive riboswitch from Escherichia coli thiM mRNA, using chemically labeled variants. Referring to a recent structure determination of the TPP/aptamer complex, each variant was synthesized with a single 2-aminopurine (AP) nucleobase replacement that was selected to monitor formation of tertiary interactions of a particular region during ligand binding in real time by fluorescence experiments. We have determined the rate constants for conformational adjustment of the individual AP sensors. From the 7-fold differentiation of these constants, it can be deduced that tertiary contacts between the two parallel helical domains (P2/J3-2/P3/L3 and P4/P5/L5) that grip the ligand's ends in two separate pockets, form significantly faster than the function-critical three-way junction with stem P1 fully developed. Based on these data, we characterize the process of ligand binding by an induced fit of the RNA and propose a folding model of the TPP riboswitch aptamer. For the full-length riboswitch domain and for shorter constructs that represent transcriptional intermediates, we have additionally evaluated ligand-induced folding via AP-modified variants and provide insights into the sequential folding pathway that involves a finely balanced equilibrium of secondary structures.
authors: Institute of Organic Chemistry, Center for Molecular Biosciences Innsbruck, Leopold Franzens University, Innrain 52a, 6020 Innsbruck, Austria.
authors: Kathrin
authors: K
authors: Lang
authors: Renate
authors: R
authors: Rieder
authors: Ronald
authors: R
authors: Micura
mesh_terms: D015075:2-Aminopurine / Q000737:chemistry; D020413:3' Untranslated Regions / Q000737:chemistry; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012329:RNA, Bacterial / Q000737:chemistry* / Q000378:metabolism; D038621:Regulatory Sequences, Ribonucleic Acid; D013050:Spectrometry, Fluorescence; D013835:Thiamine Pyrophosphate / Q000737:chemistry* / Q000378:metabolism; D014158:Transcription, Genetic; D020506:Untranslated Regions / Q000737:chemistry*
chemical_list: D020413:3' Untranslated Regions; D008024:Ligands; D012329:RNA, Bacterial; D038621:Regulatory Sequences, Ribonucleic Acid; D020506:Untranslated Regions; D015075:2-Aminopurine; D013835:Thiamine Pyrophosphate
references: Nat Rev Mol Cell Biol. 2004 Jun;5(6):451-63
references: 15173824
references: Nucleic Acids Res. 2006;34(22):6587-604
references: 17135184
references: Nucleic Acids Res. 2007;35(11):3713-22
references: 17517779
references: Nature. 2002 Oct 31;419(6910):952-6
references: 12410317
references: RNA. 2007 Apr;13(4):573-82
references: 17307816
references: Angew Chem Int Ed Engl. 2002 Jul 2;41(13):2265-9
references: 12203559
references: Tetrahedron. 2007 Apr 23;63(17):3567-6574
references: 18431442
references: Science. 2006 Oct 13;314(5797):300-4
references: 17038623
references: Nat Biotechnol. 2006 Dec;24(12):1558-64
references: 17160062
references: Curr Opin Struct Biol. 2004 Jun;14(3):344-9
references: 15193315
references: J Am Chem Soc. 2005 Aug 31;127(34):12035-45
references: 16117544
references: Chem Biol. 2006 Aug;13(8):857-68
references: 16931335
references: J Mol Biol. 2006 Jun 9;359(3):754-68
references: 16650860
references: Trends Biochem Sci. 2004 Jan;29(1):11-7
references: 14729327
references: Structure. 2006 Sep;14(9):1459-68
references: 16962976
references: ACS Chem Biol. 2006 Jul 21;1(6):341-5
references: 17163768
references: Nature. 2005 Dec 22;438(7071):1105-15
references: 16372000
references: Proc Natl Acad Sci U S A. 2001 Jan 2;98(1):37-41
references: 11120885
references: Science. 2006 May 26;312(5777):1208-11
references: 16675665
references: Biochemistry. 2006 Jun 20;45(24):7563-73
references: 16768452
references: FEBS Lett. 2003 Dec 18;555(3):516-20
references: 14675766
references: J Mol Biol. 2003 Jan 17;325(3):421-31
references: 12498793
references: Chembiochem. 2007 May 25;8(8):896-902
references: 17440909
references: Biochemistry. 2005 Oct 11;44(40):13404-14
references: 16201765
references: FEBS Lett. 2005 May 9;579(12):2583-8
references: 15862294
references: J Am Chem Soc. 2007 Mar 21;129(11):3257-66
references: 17319662
references: RNA. 2003 Jun;9(6):644-7
references: 12756322
references: Nature. 2007 May 24;447(7143):497-500
references: 17468745
references: Angew Chem Int Ed Engl. 2007;46(8):1212-9
references: 17226886
references: Tetrahedron. 2007 Apr 23;63(17):3575-3584
references: 18431441
references: Mol Cell. 2005 Apr 1;18(1):49-60
references: 15808508
references: Chembiochem. 2003 Oct 6;4(10):1024-32
references: 14523920
references: Nature. 2006 Jun 29;441(7097):1167-71
references: 16728979