title: A remarkably stable kissing-loop interaction defines substrate recognition by the Neurospora Varkud Satellite ribozyme.
abstract: Kissing loops are tertiary structure elements that often play key roles in functional RNAs. In the Neurospora VS ribozyme, a kissing-loop interaction between the stem-loop I (SLI) substrate and stem-loop V (SLV) of the catalytic domain is known to play an important role in substrate recognition. In addition, this I/V kissing-loop interaction is associated with a helix shift in SLI that activates the substrate for catalysis. To better understand the role of this kissing-loop interaction in substrate recognition and activation by the VS ribozyme, we performed a thermodynamic characterization by isothermal titration calorimetry using isolated SLI and SLV stem-loops. We demonstrate that preshifted SLI variants have higher affinity for SLV than shiftable SLI variants, with an energetic cost of 1.8-3 kcal/mol for the helix shift in SLI. The affinity of the preshifted SLI for SLV is remarkably high, the interaction being more stable by 7-8 kcal/mol than predicted for a comparable duplex containing three Watson-Crick base pairs. The structural basis of this remarkable stability is discussed in light of previous NMR studies. Comparative thermodynamic studies reveal that kissing-loop complexes containing 6-7 Watson-Crick base pairs are as stable as predicted from comparable RNA duplexes; however, those with 2-3 Watson-Crick base pairs are more stable than predicted. Interestingly, the stability of SLI/ribozyme complexes is similar to that of SLI/SLV complexes. Thus, the I/V kissing loop interaction represents the predominant energetic contribution to substrate recognition by the trans-cleaving VS ribozyme.
authors: Département de Biochimie et Médecine Moléculaire, Université de Montréal, C.P. 6128, Succursale Centre-Ville, Montréal, QC, Canada H3C 3J7.
authors: Patricia
authors: P
authors: Bouchard
authors: Département de Biochimie et Médecine Moléculaire, Université de Montréal, C.P. 6128, Succursale Centre-Ville, Montréal, QC, Canada H3C 3J7.
authors: Pascale
authors: P
authors: Legault
mesh_terms: D020029:Base Pairing; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D020134:Catalytic Domain / Q000235:genetics; D004722:Endoribonucleases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008274:Magnesium / Q000737:chemistry; D008969:Molecular Sequence Data; D009491:Neurospora / Q000201:enzymology* / Q000235:genetics; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D020871:RNA Stability / Q000235:genetics; D016337:RNA, Catalytic / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D013379:Substrate Specificity; D013816:Thermodynamics
chemical_list: D016337:RNA, Catalytic; D004722:Endoribonucleases; C438195:varkud satellite ribozyme; D008274:Magnesium
keywords: Neurospora VS ribozyme; U-turn; isothermal titration calorimetry; kissing-loop interaction; substrate recognition