A remarkably stable kissing-loop interaction defines substrate recognition by the Neurospora Varkud Satellite ribozyme

作者：Bouchard Patricia; Legault Pascale^*

来源：RNA-A Publication of the RNA Society, 2014, 20(9): 1451-1464.

DOI：10.1261/rna.046144.114

• Neurospora VS ribozyme
• substrate recognition
• U-turn
• kissing-loop interaction
• isothermal titration calorimetry

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 SE] 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 Sly stem loops. We demonstrate that preshifted SE] 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.

• 出版日期2014-9