Ligand-induced folding of the thiM TPP riboswitch investigated by a structure-based fluorescence spectroscopic approach.
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.
Lang
Kathrin
K
Institute of Organic Chemistry, Center for Molecular Biosciences Innsbruck, Leopold Franzens University, Innrain 52a, 6020 Innsbruck, Austria.
Rieder
Renate
R
Micura
Ronald
R
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*
D020413:3' Untranslated Regions; D008024:Ligands; D012329:RNA, Bacterial; D038621:Regulatory Sequences, Ribonucleic Acid; D020506:Untranslated Regions; D015075:2-Aminopurine; D013835:Thiamine Pyrophosphate
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