Potassium ions modulate a G-quadruplex-ribozyme's activity.
Hepatitis delta virus ribozyme folds into a tightly packed tertiary structure. However, unlike other ribozymes, it does not appear to be able to follow alternative folding pathways. Molecular engineering of the hepatitis delta virus ribozyme led to the development of a ribozyme possessing an endoribonuclease activity that is under the control of a G-quadruplex structure (i.e., a G-quartzyme). This latter species represents an entirely new class of ribozyme. Mutants of this ribozyme were then generated in order to shed light on the modulation of the cleavage activity caused by the presence of the G-quadruplex structure. Kinetic characterization of the G-quartzyme was performed under various single turnover conditions. It was found to be active only in the presence of potassium cations that act as counter ions in the positioning of the four coplanar guanines that form the building block of the G-quadruplex structure. The G-quartzyme behaves as an allosteric ribozyme, with the potassium cations acting as positive effectors with a Hill coefficient of 2.9 +/- 0.2. The conformation transition caused by the presence of the potassium ions is supported by enzymatic and chemical probing of both the inactive (off) and active (on) structures. This study shows that it is possible to interfere with the tight structure of the hepatitis delta virus ribozyme by adding an unusual, stable structure. To our knowledge, the G-quartzyme is the sole ribozyme that exhibits a monovalent cation-dependent activity.
Beaudoin
Jean-Denis
JD
RNA Group/Groupe ARN, Département de Biochimie, Université de Sherbrooke, Sherbrooke QC, J1H 5N4, Canada.
Perreault
Jean-Pierre
JP
D000494:Allosteric Regulation; D002414:Cations, Monovalent / Q000737:chemistry; D054856:G-Quadruplexes; D005818:Genetic Engineering; D003698:Hepatitis Delta Virus / Q000201:enzymology; D009690:Nucleic Acid Conformation; D011188:Potassium / Q000737:chemistry*; D016337:RNA, Catalytic / Q000737:chemistry* / Q000235:genetics
D002414:Cations, Monovalent; D016337:RNA, Catalytic; D011188:Potassium